Method for determining the methylation pattern of a polynucleic acid

ABSTRACT

Particular aspects relate to a method for determining the methylation pattern of a polynucleic acid, comprising: a) preparing a solution comprising a mixture of fragments of the polynucleic acid; b) coupling the fragments with a substance being detectable with a detection method; c) contacting a solution comprising the fragments of b) with a DNA microarray having a plurality of different immobilized oligonucleotides, each comprising at least one methylation site, at respectively assigned different locations thereon, the contacting under conditions affording hybridization of fragments with correlated immobilized oligonucleotides under defined stringency, and wherein the immobilized oligonucleotides have a length of less than 200 bases; d) optionally performing a a washing step; and e) detecting, using the physical detection method, such immobilized nucleic acids to which solution fragments are hybridized and/or to which solution fragments are not hybridized.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalPatent Application Nos. 60/710,556, filed 23 Aug. 2005; 60/735,349,filed 10 Nov. 2005; and 60/771,350, filed 7 Feb. 2006, and additionallyclaims the benefit of priority to German Patent Application Nos: DE102005007185.6, filed 16 Feb. 2005; DE 102005023055.5, filed 13 May2005; DE 102005025240.0, filed 31 May 2005; and DE 102005036500.0, filed28 Jul. 2005, all of which are incorporated by reference herein in theirentireties.

FIELD OF THE INVENTION

Aspects of the present invention relate generally to determining themethylation pattern of a polynucleic acid.

BACKGROUND

Many diseases, in particular cancer diseases, are accompanied bymodified gene expression. This may be related to a mutation of the genesthemselves, which leads to an expression of modified proteins or to aninhibition or over-expression of the proteins or enzymes. A modulationof gene expression may, however, also occur by epigenetic modifications,and in particular by DNA methylation. Such epigenetic modifications donot alter the actual DNA coding sequence, but nonetheless havesubstantial health implications, and it is clear that knowledge aboutmethylation processes and modifications of methylation relatedmetabolism and DNA methylation are essential for understanding,prophylaxis, diagnosis and therapy of diseases.

The precise control of genes, which themselves represent but a smallpart of the complete mammalian genome, is a question of regulation inconsideration of the fact that the bulk of genomic DNA in non-coding.The existence of such non-coding ‘trunk’ DNA containing introns,repetitive elements and potentially actively transposable elements,necessitates effective mechanisms for their durable suppression(silencing). Cytosine methylation by S-adenosylmethionine(SAM)-dependent DNA methyltransferases, which forms 5-methylcytosine,represents one such mechanism for modification of DNA-proteininteractions. Genes can be transcribed by methylation-free promoters,even when adjacent transcribed or non-transcribed regions are widelymethylated. This permits the use and regulation of promoters offunctional genes, whereas the trunk DNA including the transposableelements is suppressed. Methylation is also involved in the long-termsuppression of X-linked genes, and may lead to either a reduction or anincrease of the degree of transcription, depending on where themethylation in the transcription unit occurs.

Nearly the complete natural DNA methylation in mammals is restricted tocytosine-guanosine (CpG) dinucleotide palindrome sequences, which arecontrolled by DNA methyl transferases. CpG dinucleotides represent about1 to 2% of all dinucleotides and are concentrated in so-called CpGislands. A generally accepted definition of CpG islands means a DNAregion of about 200 bp having a CpG content of at least 50%, and wherethe ratio of the number of observed CG dinucleotides and the number ofthe expected CG dinucleotides is larger than 0.6 (Gardiner-Garden, M.,Frommer, M. (1987) J. Mol. Biol. 196, 261-282). Typically, CpG islandshave at least 4 CG dinucleotides in a sequence having a length of 100base pairs.

If CpG islands are present in promoter areas, they often have aregulatory function for the expression of the respective gene. If theCpG island is hypomethylated, expression can take place.Hypermethylation often leads to the suppression of the expression. Inthe normal state, a tumor suppressor gene is hypomethylated. If ahypermethylation takes place, this will lead to a suppression of theexpression of the tumor suppressor gene, which is frequently observed incancer tissues. In contrast thereto, oncogenes are hypermethylated inhealthy tissue, whereas in cancer tissue they are frequentlyhypomethylated.

Cytosine methylation typically prevents the binding of proteinsregulating transcription. This leads to a modification of associatedgene expression. In the context of cancer, for example, the expressionof cell division regulating genes is thereby affected (e.g., theexpression of apoptosis genes is down-regulated, whereas oncogeneexpression is up-regulated). DNA hypermethylation also has a long-terminfluence on gene regulation. Via cytosine methylation, histonede-acetylation proteins can bind to the DNA by their 5-methylcytosine-specific domain. Consequently, histones are de-acetylated,leading to a tighter DNA compaction, whereby regulatory proteins areprecluded from DNA binding.

Consequently, the efficient detection of DNA methylation patterns is animportant tool for developing new approaches for prevention, diagnosisand treatment of diseases and for target screening. In particular,individualized methylation profiles can be prepared, and a tailoredtherapy thereby deduced. Additionally, the effects of a therapy can bemonitored.

There is, therefore, a pronounced need in the art for novel andefficient methods for identifying and characterizing unknown methylationpatterns.

Differential Methylation Hybridization (DMH; Huang et al, Hum Mol Genet,8:459-470, 1999; U.S. patent application Ser. No. 09/497,855, bothincorporated by reference in their entirety) is an art-recognized methodfor determining methylation patterns or for determining hypermethylatedCpG islands. In DMH applications, DNA fragments obtained by digestionwith restriction enzymes are hybridized on a DNA microarray that carriescloned CpG islands. DNA, originating from a tissue sample, is initiallycut with a single non-methylation-specific restriction enzyme (e.g.,MseI). The resulting fragments are then ligated with linkers, and thelinker-ligated fragment mixture is cut with methylation-specificendonucleases (e.g., BstUI and/or HpaII), and amplified by means of PCR.The resulting amplified fragment mixtures are also referred to herein asDMH ‘amplificates’ or ‘amplicons.’ After a purification step, theamplicons (amplificates) are coupled with a fluorescence dye. Typically,the preceding steps are performed on the one hand with diseased tissueDNA and on the other hand with DNA from adjacent healthy tissue of thesame tissue type, and the respective fragments are labeled withdifferent fluorescence dyes. Both fragment solutions are thenco-hybridized on a DNA microarray having immobilized CpG islandsequences. After washing steps, a picture of the DNA microarray is takenwith a commercial scanner that is sensitive to fluorescence radiation.The picture or pattern of fluorescent dots visible therein is analyzedto determine differences in methylation between and among CpG clones(see, e.g., Wei et al, Clinical Cancer Research, 8:2246-2252, 2002; Yanet al, Cancer Res. 61:8375-80, 2001; see also WO 2003/087774(PCT/US03/11598), and U.S. Pat. No. 6,605,432).

In DMH applications, the immobilized nucleic acids are composed ofclones from so-called “CpG island libraries (CGI libraries)”; that is,from libraries of clones having typical lengths of 200-700 base pairsand being enriched for CpG islands. Typically, clones including repeatsequences are also present (see, e.g., WO 2003/087774 (PCT/US03/11598)).Unfortunately, the relatively high production expenses of the CGI clonelibraries are an inherent drawback of the method.

Additionally, to a significant extent the utility of DMH is limited togeneral genome analysis (discovery analysis), where only a broadanalysis of the the genome sequence is desired. This is because of: (i)the number of coupling positions on the microarray is limited; (ii) thepresence of repeat sequences unfortunately reduces the capacity of theDNA microarray; and (iii) the limited number of coupling positions onthe microarray is therefore not used in an optimum manner by differentpartial sequences.

Further drawbacks of DMH are that: sequences may be redundantly presentin CGI clone libraries; that cross contamination of the clones leads toa mixing of the library; and the possibility of cross-hybridizations,and the large expenses for production. Sequence redundancy can beexplained by the presence of partially overlapping clones, or bymultiple recurrences of the same clone. Additionally, because of thelength of the clones, the possibility of cross-hybridization eventscannot be excluded, and with increasing length, the probability ofrepeats becomes higher. The large ‘production expenses’ are caused,among other factors, by the necessity to sequence all clones of thelibrary.

A further problem in DMH applications is that the number of fragments tobe tested is enormously complex, leading to unstable signals, increasedcross-hybridization and increased occurrence of non-specifichybridization. The theoretical reason for the high complexity relates tothe fact that, in the art-recognized DMH method, all fragments that arenot cut by methylation-specific restriction enzymes are amplified in thelast step. Because the number of fragments that simultaneously have arestriction recognition sequence and are down-methylated is very small,the complexity of the mixture is extremely high, and effectivelyreflects amplification of a substantial portion of the entire genome.Therefore, a specific reduction of fragment complexity would beparticularly desirable here, because a very large number of differentfragments leads to comparatively small amplification factors; that is,individual fragments per se are only slightly amplified, and thedifference in the copy-number between methylated and unmethylatedfragments is small. Even if the amplification factor could be increased,detection of individual fragments from a very large population ofdifferent fragments would not be possible or would be substantiallyproblematic, because of cross hybridization effects. With regard to suchexcessive complexity, reference is made to the document Lucito, et al.,Genetic Research (2000).

There is, therefore, a pronounced need in the art for more simplifiedmethods to effectively reduce the complexity of the obtained DNAfragment solutions obtained in DMH applications, and preferably wheresuch methods simultaneously afford obtaining potentially interestingfragments.

A method referred to as “MSO,” has also been described by Gitan, et al(Gitan R. S., Shi H., Yan P. S., Huang T. H-M., Methylation-specificoligonucleotide microarray: A new potential for high-throughputmethylation analysis. Genome Res., 12:158-164, 2001). The Gitanimplementation describes the investigation of methylation sites within adefined region, such as a specific CpG island.

The drawbacks of methods based on analysis of bisulfite-transformed DNAare the additional expenses for the method, and the relatively high lossof DNA that occurs during the bisulfite treatment. Further, the designof the requisite oligos becomes more difficult, because the complexityof the investigated nucleic acids became less by the substantialelimination of the cytosines (by conversion of the unmethylatedcytosines into thymines).

Furthermore, the detection of SNPs (single nucleotide polymorphisms) isconsiderably more difficult and sensitive for/vulnerable to crosshybridizations.

In other contexts, microarrays carrying oligonucleotides are inprinciple known, and these oligonucleotides can be synthesized on thesubstrate of the microarray, which makes this kind of detectiongenerally advantageous for high-throughput methods.

SUMMARY OF ASPECTS OF THE INVENTION

Particular aspects provide an efficient method for identifying unknownmethylation patterns that is more effective and powerful than prior artmethods

Additional aspects provide simplified methods to more effectively reducethe complexity of the DNA fragment solutions obtained in DMHapplications, while simultaneously providing for obtaining potentiallyinteresting fragments.

Particular aspects provide a method for detecting methylationdifferences, which on the one hand permits the use of genomic DNA andneed not be based on a previous transformation such as the bisulfitetreatment, and on the other hand simultaneously affords investigation ofas many different CpG positions (CG dinucleotides) as possible, wherethe employed DNA microarray is optimized with regard to the complexityfor a comprehensive methylation analysis, and is adapted to the stepsdistinguishing the methylation patterns.

In particular aspects, the inventive methods reduce the complexity ofthe fragment mixture in the DMH method and thus lead to a significantlyincreased efficiency of the DMH method. This reduction of complexity maybe achieved in different ways: on the one hand by using at least onemethylation-specific restriction enzyme without previous addition of anon-methylation-specific restriction enzyme and subsequent amplificationof fragments of a certain size range; and on the other hand by using atleast two non-methylation-specific restriction enzymes in a first step,and after an amplification step by using at least onemethylation-specific restriction enzyme in a second digestion step.

Further, oligoarrays may be used for the method according to theinvention, in lieu of the known CpG island arrays. This leads toadditional advantages:

-   -   i) by an “in silico” definition of the oligonucleotides, regions        of the genome with repeats can be excluded, and thereby at last        the capacity of the microarray can be optimized;    -   ii) by using oligonucleotides on the microarray, the microarray        can be prepared at little cost, namely by direct synthesis on        the chip surface;    -   iii) by using oligonucleotides on the microarray, a higher        flexibility of the design and higher densities are achieved;    -   iv) by using oligonucleotides on the array, methylation        differences can still be detected in regions that have a CG        density of only 2% (conventional DMH methods typically detect        CpG islands, which have a CG density of at least 4%;    -   v) by using oligonucleotides on the array, it is possible to        examine a substantially larger number of potentially interesting        methylation sites, than this was possible with a CGI clone        array, and thereby the whole genome can be tested for different        methylation in a single hybridization step with a corresponding        array;    -   vi) because the sequence of the oligonucleotides is known, and        they are specifically synthesized, sequencing as in the case of        the clones of a conventional array is not necessary;    -   vii) because the sequence of the oligonucleotides is known, and        they are specifically synthesized, a redundancy (as in        conventional arrays) of the chips can be prevented; and    -   viii) by using oligonucleotides that are only up to 80 bp long,        cross hybridization events (a problem with conventional array)        are effectively excluded.

Oligoarrays may also be used in a combination with other non-DMH methodsfor producing fragments. In particular, it is possible to specificallyenrich methylated or unmethylated fragments, and to then analyze them bymeans of oligonucleotide arrays. The use of oligonucleotide arrays indiscovery applications leads to many advantages compared to the priorart. A method for determining methylation patterns, which usesimmobilized oligonucleotides in lieu of immobilized clones has beenpreviously described. However, amplification of the nucleic acids beforehybridization thereof with these oligo arrays is a requirement of suchmethods. Therefore, such oligos, typically used in pairs, are onlysuitable to detect methylation in converted/treated/transformed nucleicacids (e.g., subjected to bisulfite treatment). In such applications,the epigenetic difference (is there a methyl group at the cytosine ornot) becomes obvious only after the treatment, such that it will bemaintained and thus detectable as a sequence difference (thymines orcytosines) after an amplification (e.g., by PCR). The methylation degreein the tested and amplified sample can then be determined by usingCG-specific and TG-specific oligos. This technology is, for example,described in more detail in WO 01/38565 (U.S. Ser. No. 10/148,140) andWO 02/18632 (U.S. Ser. No. 10/363,345).

Particular aspects provide a method for determining the methylationpattern of a polynucleic acid, comprising:

a) obtaining a solution comprising a nucleic acid, and obtainingtherefrom a solution comprising a mixture of fragments of thepolynucleic acid, wherein the composition of the fragment mixturedepends on the methylation pattern of the polynucleic acid;

b) the fragments of step a) are optionally amplified and coupled with asubstance being detectable with an optionally physical detection method,wherein optionally an amplification of the fragments may occur;

c) a solution comprising the fragments of step b) is contacted with aDNA microarray having a plurality of different immobilized nucleicacids, in particular oligonucleotides, under conditions wherehybridization of fragments occurs with correlated immobilized nucleicacids under a defined stringency,

wherein the immobilized nucleic acids are selected from nucleic acids,which are specific for fragments of a genome, preferably the humangenome, and are localized on the DNA microarray at differentrespectively assigned positions on the DNA microarray;

e) optionally, a washing step is performed;

d) such immobilized nucleic acids, to which fragments of the solutionare hybridized and/or to which fragments of the solution are nothybridized, are detected using the detection method,

f) optionally, from the detected hybridizations and/or detectednon-hybridizations according to step e) the methylation pattern of thepolynucleic acid being the educt of step a) is derived.

Further, aspects relate to: a test kit for performing one of the abovemethods; a method for preparing such a DNA microarray suitable fordetermining the methylation pattern of a polynucleic acid; the use ofsuch a method for determining the methylation pattern of a polynucleicacid for identifying an indication-specific marker or a target or amodulator for such a target, the use of such a modulator for preparing apharmaceutical composition having the specific indication, and the useof such a method or test kit for diagnosing and/or prognosticating adisease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the fragment length distribution for fragments withoutmethylation-specific restriction sites in broken lines. The continuouslines represent the fragment length distribution for fragments withmethylation-specific restriction sites. The latter are particularlyinteresting for the further analysis. FIG. 1 shows the resultssubstantially according to the prior art, using anon-methyltion-specific restriction enzyme (MseI) and twomethylation-specific restriction enzymes (BstU1, HapII).

FIG. 2 shows the fragment length distribution for fragments withoutmethylation-specific restriction sites in broken lines. The continuouslines represent the fragment length distribution for fragments withmethylation-specific restriction sites. FIG. 2 shows the results of themethod according to the invention, i.e. using severalnon-methylation-specific restriction enzymes, namely MseI, Bfa1 andCsp6, and several methylation-specific restriction enzymes, namelyBstU1, HapII, HpyCH4IV and HinP1.

FIG. 3 shows an Illustration of the method for preparing DMHamplificates. Modifications relative to Huang et al. are indicated. FIG.3 illustrates the differences of the working procedures (workflow) ofthe preparation of a mixture of methylated fragments of a samplecontaining DNA.

FIG. 4 shows Intra-workflow and inter-workflow reproducibilityCorrelation plots comparing the hybridization signal (averagedintensities of the log₂ signal of all detection oligomers of a fragment)of four arrays, and DNA from breast cancer cell lines was used. Thereproducibility of the complete DMH amplicon preparation (inter-workflowreproducibility, red box) and the preparation of the hybridizationsamples (starting with the adaptor mediated PCR, step 4, see FIG. 1) ofeach DMH amplicon (intra-workflow reproducibility, green boxes) wasdetermined.

FIG. 5 shows selection of marker candidates and reproducibility. A)Distribution of log₂ ration against the mean hybridization signalintensities of all fragments of the DMH amplicon generated from PBL andbreast cancer cell line DNA. Dots above and below the red lines indicatepotential marker candidates being methylated (log ratio<−0.5) orunmethylated (log ratio>0.5) in breast cancer cell line compared to PBL.B) Correlation plot of two DMH discovery experiments using PBL andbreast cancer cell line samples. Dots in the red boxes indicatepotential marker fragments reproducibly found in both experiments to beeither methylated or unmethylated in breast cancer cell lines comparedto PBL.

FIG. 6 shows validation of the technology by direct bisulfitesequencing. Validation of marker candidates found by an optimized DMHworkflow and an array hybridization. A) Examples of direct bisulfitesequencing results generated by analysis by the proprietary ESMEsoftware (Joern, L. et al; 2004). Yellow and blue indicate unmethylatedand methylated CpG's, respectively. B) Correlation of methylation stateof the 111 analyzed fragments determined by bisulfite sequencing withlog ratio as determined by DMH.

FIG. 7 shows fragment length distribution after “in silico” digestionwith BstU for DNA sections with a share of CpG islands of more than 0.3.FIG. 8 shows a fragment/length histogram after “in silico” digestionwith BstU for DNA sections with a share of CpG islands of at most 0.3.

FIG. 9 shows boxplots of signal intensity values as presented in Table 3(y-axis) for three microarray-chips (x-axis, chips 1, 2, 3).

FIG. 10 shows boxplots of signal intensity values as presented in Table4 (y-axis) after “log₂ transformation” for the same threemicroarray-chips as FIG. 9 (x-axis, chips 1, 2, 3).

FIG. 11 shows boxplots of signal intensity values as presented in Table8 (y-axis) after “log₂ transformation” and “Quantile Normalization” forthe same three microarray-chips as FIG. 9 (x-axis, chips 1, 2, 3).

FIG. 12 shows boxplots of signal intensity values as presented in Table9 after “log₂ transformation”, “Quantile Normalization” and “Baselineshift” for the same three microarray-chips as FIG. 9 (x-axis, chips 1,2, 3).

FIG. 13 shows boxplots of signal intensity values as presented in Table10 after “log₂ transformation”, “Quantile Normalization”, “Baselineshift” and generation of representative values by selecting a medianvalue for the same three microarray-chips as FIG. 9 (x-axis, chips 1, 2,3).

DETAILED DESCRIPTION

For achieving various technical objects, particular aspects of theinvention teach and provide a method for determining the methylationpattern of a polynucleic acid with the following steps:

a) a solution comprising a mixture of fragments of the polynucleic acidis made from a solution comprising the polynucleic acid, e.g. a solutioncomprising genomic DNA, wherein the composition of the fragment mixturedepends on the methylation pattern of the polynucleic acid,

b) the fragments of step a) are coupled with a substance beingdetectable with an optionally physical detection method, whereinoptionally an amplification of the fragments may occur,

c) a solution comprising the fragments of step b) is contacted with aDNA microarray, which carries a plurality of different immobilizednucleic acids, in particular oligonucleotides, under conditions, atwhich a hybridization of fragments occurs with correlated immobilizednucleic acids under defined stringency,

wherein the immobilized nucleic acids are selected from nucleic acids,which are specific for fragments of a genome, preferably the humangenome, and are localized on the DNA microarray at differentrespectively assigned positions,

d) optionally, a washing step is performed;

e) such nucleic acids, to which fragments of the solution are hybridizedand/or to which fragments of the solution are not hybridized, aredetected using the detection method;

f) optionally, from the detected hybridizations and/ornon-hybridizations according to step e) the methylation pattern of thepolynucleic acid being the educt of step a) is derived.

Significantly, the method according to the invention may be performed inparticular without a bisulfite treatment in step a) or before step a).

I. First Preferred Embodiment Complexity Reduction by UsingMethylation-Specific Restriction Enzymes

A first particularly preferred embodiment of a method according to theinvention for achieving the technical object provides a method formaking a mixture of fragments of a polynucleic acid, comprising:

a) a solution comprising a polynucleic acid is prepared;

b) optionally, a processing step is performed, in which substances thatare not polynucleic acids, are depleted, or the polynucleic acid isenriched;

c) a methylation-specific restriction enzyme is added to the solution inthe absence of prior addition of (e.g., digestion with) anon-methylation-specific restriction enzyme, wherein the polynucleicacid is cut to fragments at cutting sites, which are capable of beingmethylated, but are not methylated; and

d) the fragments obtained in step c) are subjected to an amplificationstep after adapter ligation, and fragments having a length in the rangefrom 50 bases to 5,000 bases are selectively enriched.

In an alternate embodiment of the invention, fragments afteradapterligation are subject to a digestion with the same restrictionenzyme or enzymes as used in c) before amplification. This has theadvantage that religated fragments are digested while fragment-adapterligations remain unaffected.

Therefore, in particular aspects, a solution with a mixture of fragmentsof a polynucleic acid is obtained, which is for instance suitable forDMH analysis. By not using a non-methylation-specific restriction enzymein connection with selective amplification of the specified lengthwindow, a reduction of complexity of the mixture by a factor greaterthan 100 is achievable; that is, by a factor 100 better than in priorart DMH applications/implementations. This is also based on the factthat methylated sequence regions and sequence regions without cgcgelements (recognition sites for methylation sites) are not cut, andconsequently form fragments, the length of which is usually above theupper limits of the amplification window. By contrast, regions withrecognition sites, as far as not methylated, are cut and form fragmentswith a length below the upper limit of the amplification window.Further, no potentially interesting fragments are cutnon-methylation-specifically, and thus reduced to a length below thelower limit of the window. Consequently, all interesting fragments(i.e., those with potentially hypermethylated or hypomethylated sites)are available for subsequent analyses. Finally, the overall process issimplified, because different restriction enzymes are used at a lesserdegree. It is, for example, possible to perform all reactions up to thehybridization on a DMH chip in one vessel (one tube process).Consequently, processing is simplified and considerably faster. Finally,the number of potential error sources in the process is substantiallyreduced.

It is, therefore, particularly preferred that there is not used anon-methylation-specific restriction enzyme in any of the steps.

In principle, every methylation-specific restriction enzyme can be usedfor the purpose of the invention. Preferably the methylation-specificrestriction enzyme is selected from the group consisting of BisI, BstUI,BshI236I, AccII, BstFNI, McrBC, GlaI, MvnI, HpaII (HapII), HhaI, AciI,SmaI, HinP1I, HpyCH4IV, and mixtures of two or more of said enzymes.

In particular aspects, the polynucleic acid used is a naturallyoccurring DNA. Preferably, genomic DNA is used (e.g, human genomic DNA).

Preferably, in d), fragments with a length of 80 to 5,000 bases,preferably 100 to 2,000 bases, and most preferably 100 to 1,000 bases,are selectively enriched, and optimal complexity reduction is achieved,while simultaneously preserving and/or enriching the presence ofpotentially interesting fragments.

A further complexity reduction can be achieved, if the fragments arepurified by physical methods (e.g., by gel electrophoresis, sizeexclusion chromatography, filtration, etc.) before or after theamplification.

The invention further relates to a method for determining themethylation pattern of a polynucleic acid, in particular that of agenomic DNA from a tissue sample of a patient, the method comprising:

-   -   a) a process according to one embodiment of the invention is        performed;    -   b) thereafter the selectively amplified fragments are coupled        with a substance being detectable in a physical detection        method;    -   c) a solution comprising the fragments of step b) is contacted        with a DNA microarray having a plurality of different        immobilized nucleic acids with at least one methylation site        each at different, respectively assigned locations on the        microarray, wherein a hybridization of fragments with correlated        immobilized nucleic acids takes place with defined stringency;    -   d) optionally, a washing step is performed; and    -   e) such nucleic acids, to which fragments of the solution are        hybridized, are detected using the physical detection method.

The detectable substance may, for example, be a fluorescence dye,wherein the detection method comprises selective scanning for thefluorescence radiation transmitted by the fluorescence dye(one-dimensional or two-dimensional, depending on the arrangement of thedifferent immobilized nucleic acids on the chip). The fluorescence dyemay be, for example, Cy3 and/or Cy5. As recognized by those of ordinaryskill in the relevant art, many other fluorescence dyes would besuitable for the present purposes.

The inventive methods can be used for various purposes. On the one hand,DMH applications can be carried out as explained in further detailherein below. The inventive methods can, however, also be used fordiagnostic purposes. In the latter case, the immobilized nucleic acidscontain, for example, nucleic acid sequences, the methylation sites ofwhich are not methylated compared to the normal state, if a defineddisease occurs. In this context, fragments of the tested DNA hybridizingtherewith will indicate that a disease occurs, because the fragments areexclusively those fragments that are not methylated in the tested DNA.Of course, immobilized nucleic acids may also or additionally be used,which are methylated in case of a disease. Then, by non-hybridization,exclusion information is obtained. Further, it is possible to generatethe immobilized nucleic acids by digestion with methylation-specificrestriction enzymes, which cut the DNA, if a cytosine methylationexists.

According to alternate aspects of the invention, various DNA arrays canbe used. In a preferred embodiment, arrays are used, as they are knownfrom conventional methods. In particular aspects, these arrays comprisecloned CpG islands.

In a particularly preferred embodiment, oligo chips are used, and theseare described below in more detail. Explicit reference to theseapplications is made.

For this variant, the DNA microarray may in principle carry immobilizednucleic acids, the methylation or non-methylation of which is correlatedwith a plurality of different defined diseases. Accordingly, the DNA ofthe patient is thereby simultaneously tested for the plurality ofdifferent diseases. Because of the complexity of such examinations, itis preferred that the DNA microarray carries nucleic acids containingeither exclusively methylated or exclusively not methylated sequences,compared to the normal state.

In conjunction with such variants, the invention further provides a testkit for performing the inventive method, the kit comprising: i) a singlerestriction enzyme component, which comprises exclusively amethylation-specific restriction enzyme or several of such enzymes, andii) a DNA microarray, which carries a plurality of different immobilizednucleic acids with a methylation site each at respectively assigneddifferent locations on the DNA microarray, and the nucleic acids maycontain at least one methylation site. The nucleic acids further containnucleic acid sequences, which are not methylated or are methylated for aplurality of different defined diseases or a single defined disease,compared to the normal state. The defined disease may for instance be aspecific cancer disease. A specific cancer disease is, for example, anorgan-specific cancer disease, such as lung cancer, ovary cancer,scrotal cancer, prostate cancer, pancreas cancer, cancer of an organ ofthe digestive tract, etc. Suitable sequences with regard to all aspectsof the invention are for example, described in the documents DE 20121979U1, DE 20121978 U1, DE 20121977 U1, DE 20121975 U1, DE 20121974 U1, DE20121973 U1, DE 20121972 U1, DE 20121971 U1, DE 20121970 U1, DE 20121969U1, DE 20121968 U1, DE 20121967 U1, DE 20121966 U1, DE 20121965 U1, DE20121964 U1, DE 20121963 U1, DE 20121961 U1, DE 20121960 U1, DE 10019173A1, DE 10019058 A1, DE 10013847 A1, DE 10032529 A1, DE 10054974 A1, DE10043826 A1, DE 10054972 A1, DE 10037769 A1, DE 10061338 A1, DE 10245779A1, DE 10164501 A1, DE 10161625 A1, DE 10230692, DE 10255104, EP1268855, EP 1283905, EP 1268857, EP 1294947, EP 1370685, EP 1395686, EP1421220, EP 1451354, EP 1458893, EP 1340818, EP 1399589, EP 1478784, WO2004/035803, and WO 2005/001141, all of which are incorporated byreference herein in their entirety.

In the test kit, one or several of the following components may inaddition be included: i) a linker or several linkers, if applicable in asuitable solution, ii) substances or solutions for performing a PCR,iii) a dye or several dyes, if applicable with a coupling reagent, ifapplicable in a solution, iv) substances or solutions for performing ahybridization, and/or v) substances or solutions for performing awashing step.

II. Second Preferred Embodiment The Use of AdditionalNon-Methylation-Specific Restriction Enzymes

A second particularly preferred embodiment of the inventive method forachieving the technical object, the reduction of complexity, theinvention teaches and provides a method for making a mixture offragments of a polynucleic acid, the method comprising:

a) a solution comprising a polynucleic acid is prepared;

b) optionally, a processing step is performed, in which substances thatare not polynucleic acids are depleted, and/or the polynucleic acid isenriched;

c) at least two different non-methylation-specific restriction enzymesare added to the solution, wherein the polynucleic acid is cut atrestriction sites, for which the restriction enzymes are specific;

d) fragments with a length of less than 50 bases are separated from thesolution obtained in step c);

e) linkers are ligated to the fragments obtained in step d);

f) then one or at least two methylation-specific restriction enzymes areadded to the solution obtained in step e), wherein the fragments are cutat cutting sites, which are capable of being methylated, but are notmethylated; and

g) the fragments obtained in step f) are subjected to an amplificationstep, wherein those fragments are amplified, which have not been cut instep f).

Preferably, fragments having a length in the range from 50 bases to5,000 bases are selectively enriched.

Accordingly, a solution with a mixture of fragments of a polynucleicacid is obtained, which is particularly suitable for DMH. Surprisingly,despite the increase of the number of cuts, an increase of the number ofinteresting fragments (i.e., fragments comprising CpG islands) isachieved by using several different non-methylation-specific restrictionenzymes, and the complexity (i.e., the number of the amplified fragmentsin the mixture and thus of the nucleic acids is simultaneously reduced).

Prior art methods, by contrast, have tried to reduce the complexity andto obtain interesting fragments by as few steps as possible withnon-methylation-specific restriction enzymes. By means of the instantinvention, a reduction of the complexity of the mixture (number ofamplifiable nucleic acids in the mixture) up to a factor 10 isachievable (i.e., by a factor of maximum 10 better) relative to theprior art methods.

Preferably, in step c) three different non-methylation-specificrestriction enzymes are added. It is additionally preferred, that atleast one, and preferably all non-methylation-specific restrictionenzymes cut recognition sequences having a length of four bases, and inparticular recognition sequences that do not contain CG dinucleotidesequences. Using restriction enzymes with recognition sequences having alength of four bases facilitates the generation of fragments that areshort and that are separable by purification, which reduces thecomplexity. Simultaneously, by using restriction enzymes withrecognition sequences having a length of four bases, the number ofpotentially interesting fragments (i.e., fragments, which possiblycomprise CpG islands, or amplifiable size fragments having a density ofCG dinucleotides that is increased relative to the average CG density inthe genome) is increased.

Advantageously, at least one, preferably all non-methylation-specificrestriction enzymes generate sticky ends, in particular sticky ends withan overhang containing TA. Particularly preferred is the use ofnon-methylation-specific restriction enzymes, which cut a recognitionsequence of four bases to sticky ends, and all restriction enzymesproduce the same overhangs.

Alternatively, one or several non-methylation-specific restrictionenzymes that produce sticky ends can be used in conjunction with one orseveral non-methylation-specific restriction enzymes that produce bluntends, since a ligation of a fragment with a sticky end with a fragmentwith a blunt end is also possible.

Of course it is also possible to use only non-methylation-specificrestriction enzymes that produce blunt ends, because in this case aligation is also possible.

The non-methylation-specific restriction enzymes are preferably selectedfrom at least two, and preferably three from the group consisting of“MseI, BfaI, Csp6I, Tru1I, Tvu1I, Tru9I, Tvu9I, MaeI and XspI”.Particularly preferred is the use of a combination of MseI, BfaI andCsp6I.

In principle, step c) may be performed with common (i.e., simultaneous)addition of all non-methylation-specific restriction enzymes to thesolution. Alternately, it is possible to add the restriction enzymessequentially during step c).

In principle, for step f), every methylation-specific restriction enzymecan be used. This may be enzymes, which cut methylation-specificunmethylated DNA, or enzymes, which cut methylation-specific methylatedDNA. Preferably, the methylation-specific restriction enzyme is selectedfrom the group consisting of BisI, BstUI, BshI236I, AccII, BstFNI,McrBC, GlaI, MvnI, HpaII (HapII), HhaI, AciI, SmaI, HinP1I, HpyCH4IV,EagI and mixtures of two or more of the above enzymes. Preferred is amixture containing the restriction enzymes BstUI, HpaII, HpyCH4IV andHinP1I.

In particular aspects, the inventive method is employed with apolynucleic acid that is a naturally occurring DNA. Preferably, this isa genomic DNA (e.g., a human genomic DNA).

Preferably, fragments with a length from 100 bases to 1,000 bases areselectively enriched in step e). Thereby, the optimum reduction ofcomplexity is achieved, and wherein simultaneously effectively allpotentially interesting fragments are preserved and/or enriched.

The invention further relates to a method for determining themethylation pattern of a polynucleic acid, in particular a genomic DNAfrom a tissue sample of a patient, the method comprising:

a) a method according to one embodiment of the invention is performed;

b) thereafter the selectively amplified fragments are coupled with asubstance being detectable in a physical detection method;

c) a solution comprising the fragments of step b) is contacted with aDNA-microarray, which carries a plurality of different immobilizednucleic acids with at least one methylation site each at different,respectively assigned locations, under conditions, at which ahybridization of fragments with correlated immobilized nucleic acidstakes place with defined stringency;

d) optionally, a washing step is performed;

e) such nucleic acids, to which fragments of the solution arehybridized, are detected using the physical detection method.

In such applications, the detectable substance may be a fluorescencedye, wherein the detection method then comprises selectively scanningfor the fluorescence radiation transmitted by the fluorescence dye(one-dimensional or two-dimensional, depending on the arrangement of thedifferent immobilized nucleic acids on the chip). The fluorescence dyemay, for instance, be Cy3 and/or Cy5. A person of ordinary skill in therelevant art would recognize that many other suitable fluorescence dyescould be used.

Such inventive embodiments may be used for various purposes. On the onehand, DMH can be carried out, and further applications are given anddescribed elsewhere in this application.

Alternatively, however, the inventive methods are used for diagnosticpurposes. In the latter case, the immobilized nucleic acids contain, forexample, nucleic acid sequences, the methylation sites of which are notmethylated compared to the normal state, if a defined disease occurs.Accordingly, fragments of the tested DNA hybridizing therewith willindicate that a disease does not exist, because the fragments areexclusively those fragments that are methylated in the tested DNA (andtherefore not cut, and are consequently amplifiable). Of course,immobilized nucleic acids may also or additionally be used, which arenot methylated in case of a disease. Then, by non-hybridization,exclusion information is obtained.

In various aspects, different DNA arrays can be used. In a preferredembodiment, arrays are used, as they are known from conventionalmethods. In particular aspects, these arrays comprise cloned CpGislands.

In a particularly preferred embodiment, oligo chips are used, and theseare described below in more detail. Explicit reference to theseapplications is made.

For this variant, the DNA microarray may in principle carry immobilizednucleic acids, the methylation or non-methylation of which is correlatedwith a plurality of different defined diseases. Accordingly, the DNA ofthe patient is simultaneously tested for the plurality of differentdiseases. Because of the complexity of such examinations, it ispreferred that, with respect to each single defined disease, the DNAmicroarray exclusively carries nucleic acids containing either nucleicacid sequences that are methylated, or not methylated compared to thenormal state for any single defined disease. Correspondingconsiderations apply for the case of the examination for response.

In conjunction with such variants, the invention further provides a testkit for performing the inventive method, the kit comprising: i) a firstrestriction enzyme component, which comprises at least two differentnon-methylation-specific restriction enzymes; ii) a second restrictionenzyme component, which comprises exclusively a methylation-specificrestriction enzyme or several of such enzymes; and iii) a DNAmicroarray, which carries a plurality of different immobilized nucleicacids at respectively assigned different locations on the DNAmicroarray, and the nucleic acids may comprise at least one methylationsite. Preferably, the nucleic acids comprise nucleic acid sequencesthat, for a plurality of different defined diseases or a single defineddisease, are either not methylated, or are methylated compared to thenormal state. The defined disease may for instance be a specific cancerdisease.

The test kit, may further comprise one or more of the following: i) alinker or several linkers, optionally in a suitable solution; ii) aligase, optionally in a suitable solution; iii) substances or solutionsfor performing a PCR; iv) a dye or several dyes, optionally with acoupling reagent, optionally in a solution; v) substances or solutionsfor carrying out a hybridization; and/or vi) substances or solution forcarrying out a washing step.

III. Third Particularly Preferred Embodiment Combination of ComplexityReduction and Use of Oligochips

In both above embodiments, it is particularly preferred that oligochipsare used. For achieving the technical object, the invention teaches amethod for determining the methylation pattern of a polynucleic acid onthe basis of an oligochip with the following steps:

a) a solution comprising a mixture of fragments of the polynucleic acidis made from a solution comprising a polynucleic acid (e.g. a solutioncomprising genomic DNA) using a non-methylation-specific restrictionenzyme or several non-methylation-specific restriction enzymes and afteradapter ligation, optionally using a methylation-specific restrictionenzyme or a selection of methylation-specific restriction enzymes,wherein the composition of the fragment mixture depends on themethylation pattern of the polynucleic acid;

b) the fragments are amplified and coupled with a substance beingdetectable with an optionally physical detection method;

c) a solution comprising the fragments of step b) is contacted with aDNA microarray, which carries a plurality of different immobilizedoligonucleotides, under conditions, at which a hybridization offragments occurs with correlated immobilized nucleic acids under definedstringency,

wherein the immobilized nucleic acids are selected from nucleic acids,which are specific for fragments of a genome, preferably the humangenome, of a gene bank, wherein the fragments of the genome areobtainable by means of the restriction enzymes used in step a), and arelocalized on the DNA microarray at different respectively assignedpositions;

d) optionally, a washing step is performed;

e) such immobilized nucleic acids, to which fragments of the solutionare hybridized and/or to which fragments of the solution are nothybridized, are detected using the detection method;

F) optionally, from the detected hybridizations and/ornon-hybridizations according to step E) the methylation pattern of thepolynucleic acid being the educt of step A) is derived.

Significantly, the method according to the invention may be performedwithout a bisulfite treatment in step a) or before step a).

In a preferred embodiment, the immobilized nucleotides have a length ofless than 200 bases. Therefore, in the following they are referred to asoligonucleotides.

By using oligonucleotides on the microarray, the microarray can beproduced at relatively low expenses, namely by direct synthesis on thechip surface. The oligonucleotides may however also be prepared outsidethe chip surface, and may then be applied to the chip surface by avariety of art-recognized means. This procedure has the advantage thatthe identity and quality of the oligonucleotides can be investigatedbefore using them, which will lead to very reproducible experiments. Onthe other hand, by this method, the same densities cannot be obtained asby the synthesis on the chip. In total, immediate advantages of theoligonucleotide arrays result thereby compared to the DNA array typesused in the literature, not to speak of the flexibility of the design.

For oligo chip preparation, initially the desired different sequences ofthe oligonucleotides have to be defined. A spot on the substrate of themicroarray is then assigned to each of the defined oligonucleotides.Then, at such spots, optionally the synthesis of the associatedoligonucleotide is performed. It is in particular advantageous that bythe preferred method according to the invention, the definition of thedesired oligonucleotides can be made “in silico”. In particular, regionsof the genome with repeats can be excluded when defining the sequencesof the oligonucleotides, and thus at last the capacity of the microarraycan be optimized.

Further, in the context of current commercially availableoligonucleotide microarrays or oligonucleotide chips, it is possible toexamine a substantially larger number of potentially interestingmethylation sites, than this was possible with a CGI clone array. Thus,by a single hybridization step with a corresponding array, nearly thefull genome can be investigated for different methylation. By using amethod for the matching oligo selection as described below, methylationdifferences can be detected by the above method even in regions, whichhave a CG density of 2% only. Therein, too, it is different from theknown DMH method typically limited to the analysis of CpG islands havinga CG density of at least 4% (Heisler L E, Torti D, Boutros P C, WatsonJ, Chan C, Winegarden N, Takahashi M, Yau P, Huang T H, Farnham P J,Jurisica I, Woodgett J R, Bremner R, Penn L Z, Der S D. CpG Islandmicroarray probe sequences derived from a physical library arerepresentative of CpG Islands annotated on the human genome (NucleicAcids Res. 33:2952-61, 2005).

The oligonucleotides preferably have a length of 15 to 80 bases, inparticular 20 to 30 bases.

The sequences of the oligonucleotides can in particular be defined bythe following steps:

a) the genome of an organism is tested for first partial sequences,which are limited by cutting sites of the used non-methylation-specificrestriction enzymes and have a length of 100 to 1,200 base pairs, andsaid first partial sequences are selected;

b) from the first partial sequences, are excluded those that containmore than 50% repeats, preferably partial sequences are excluded thatcontain more than 20% repeats, whereby a group of second partialsequences is formed, and where the steps a) and b) can be performed inany order.

For the case that methylation-specific restriction enzymes are furtherused for preparing the mixture of fragments, in a further step c), theselected second partial sequences are tested for cutting sites of themethylation-specific restriction enzymes used, and as third partialsequences those second partial sequences are selected, which containsuch cutting sites, and wherein the steps a) to c) can be performed inany order.

These partial sequences correspond to the sequences of the fragmentsobtained in step a). In a preferred embodiment, they are characterizedin that they contain at least one CG position within amethylation-specific restriction cutting site. Arbitrarily or accordingto further defined criteria, oligonucleotide sequences are therebyselected, which hybridize to these partial sequences (or their counterstrand) or are identical to them (in order to then hybridize to thecounter strand).

It is preferred that these oligonucleotides are intended for thesynthesis on the DNA microarray. The above steps can be performed bymeans of simple programs based on publicly accessible gene databases. Itis further preferred that several used oligonucleotide sequenceshybridize to a fragment to be detected. Herein it is preferred that 3 to30 oligonucleotides hybridize to a fragment. It is particularlypreferred that 5 to 25 oligonucleotides hybridize to a fragment, and itis most particularly preferred that 10 to 20 oligonucleotides hybridizeto a fragment.

In particular aspects, these oligo sequences overlap in part.

In a preferred embodiment, from among the possible oligonucleotidesequences, those are selected that have the smallest signal/noise ratioand/or the smallest cross hybridization rate.

It is preferred that the detectable substance is a fluorescence dye, andwherein the detection method comprises a scanning selective for thefluorescence radiation transmitted by the fluorescence dye,one-dimensional or two-dimensional, depending on the arrangement of thedifferent immobilized nucleic acids on the chip. The fluorescence dyemay be selected from the group consisting of “Cy3 and Cy5”. A person ofordinary skill in the relevant art is will be familiar with many othersuitable fluorescence dyes.

In another preferred embodiment, the detectable substance may howeveralso be a biotin, which in the detection method interacts with anothersubstance and is detected thereby (see e.g., “Gene Chip Mapping AssayManual” of Affymetrix Inc.). Fragments from different samples areseparately hybridized on the microarrays, since they cannot bedistinguished by the detectable substance, thus two identical arrays areneeded, which have then to be standardized for the comparativeevaluation.

The fragments detected with the immobilized oligonucleotides preferablycontain nucleic acid sequences, the methylation sites of which are notmethylated or are methylated compared to the normal state if a defineddisease occurs. It is however not necessary that the oligonucleotideitself contains this methylation site. The use of such oligonucleotidescontaining this methylation site is a alternate embodiment of themethod.

The DNA microarray may exclusively carry oligonucleotides, which detectnucleic acid sequences (by hybridization), which are not methylated orare methylated compared to the normal state if a single defined diseaseoccurs. It is however also imaginable that on a microarray, differentsets of oligonucleotides are immobilized, which can detect not onlydifferent fragments being specifically methylated for a disease, butalso different sets of fragments, which in turn are specificallymethylated for different diseases or other conditions of interest, andthus the occurrence of a plurality of diseases or other conditions ofinterest, which are characterized by a differential methylation, cansimultaneously be determined.

Other conditions of interest are, for instance, the risk to suffer froma certain disease, the prognosis of a certain type of a disease or thesusceptibility to side effects of a certain type of treatment. Alsodetectable are determinations/statements about the type or theaggressiveness or progress of a disease, for instance of a tumordisease, or about the efficiency of a therapy, if thesedeterminations/statements are based on methylation differences.

Further it is possible to simultaneously perform SNP analyses on thesame microarray by means of another oligo set, and thus to generateeither further information about conditions based on genetic differences(SNP differentiation) or about the type or the aggressiveness orprogress of a disease, for instance a tumor disease, or about theefficiency of a therapy, if these statements are based on SNPdifferences.

The invention also relates to oligonucleotide arrays, theoligonucleotides of which were selected according to the above criteria.

The invention moreover relates to a method for preparing such arrayssuitable for the methylation analysis, which is characterized by thatthe oligonucleotides immobilized on the surface of the array are subjectto a selection, which is based on the method described above.

The invention further relates to a test kit for performing a methodaccording to the invention, comprising the following components: arestriction enzyme component or several different restriction enzymecomponents, the restriction enzymes of which are suitable for preparingthe fragments, and a DNA microarray, which carries a plurality ofdifferent immobilized oligonucleotides at respectively assigneddifferent places on the DNA microarray. The oligonucleotides arecharacterized in that they are not longer than 200 bp.

In another test kit, a methylation-specific restriction enzyme isadditionally included, the oligonucleotides on the chip arecharacterized in that they hybridize to fragments, which contain arestriction cutting site of at least one of the employedmethylation-specific restriction enzymes.

The oligonucleotides on the array can specifically hybridize to fragmentsequences containing nucleic acid sequences, which are not methylated orare methylated compared to the normal state if a single defined diseaseoccurs. Thereby, the test kit would be suitable for the diagnosis of aspecific disease. The disease may be, for example, a specific cancerdisease.

One or several of the following components, which usually are used for aDMH analysis, may be additionally included: a linker or several linkers,if applicable in a suitable solution; substances or solutions forperforming a PCR; a dye or several dyes, if applicable with a couplingreagent, if applicable in a solution; substances or solutions forperforming a hybridization; and/or substances or solutions forperforming a washing step.

The preparation of the fragments of the polynucleic acid may beperformed in a variety of ways, for instance corresponding to thedocument Huang et al. (for bibliography see elsewhere in thisspecification). For instance, the following steps may be provided:

a) a solution containing the polynucleic acid is prepared;

b) as an option a processing step is performed, in which substances notbeing polynucleic acids are depleted and/or the nucleic acid isenriched;

c) one or preferably at least two different non-methylation-specificrestriction enzymes are added to the solution, the polynucleic acidbeing cut at cutting sites being specific for the restriction enzymes;

d) the solution obtained in step c) is purified while separating smallfragments;

e) linkers are ligated to the fragments obtained in step d);

f) then one or preferably at least two methylation-specific restrictionenzymes are added to the solution obtained in step e), the fragmentsobtained in step d) being cut at cutting sites, which are capable ofbeing methylated, but are not methylated;

and g) the fragments obtained in step f) are subjected after a furtherpurification step performed as an option to an amplification step, thosefragments being amplified, which were not cut in step f).\

In a preferred embodiment, in step d) of the above method, fragmentshaving a length of less than 40 bp, preferably less than 70 bp, and morepreferably less than 100 bp are separated from the solution obtained instep c).

In a preferred embodiment, the amplification in step g) takes place bymeans of primer molecules, which hybridize to the linkers introduced instep e), and of a polymerase under suitable PCR conditions.

Preferably, fragments having a length in the range from 50 bases to5,000 bases, preferably from 70 to 2,000 bases, and more preferably from100 to 1,200 bases are thus selectively enriched.

Thereby, a solution with a mixture of fragments of a polynucleic acid isobtained, which is particularly suitable for the method according to theinvention. For this method, it is particularly preferred that in step c)at least two non-methylation-specific restriction enzymes are used. Inthe case of at least two non-methylation-specific restriction enzymes,on the one hand the number of those fragments is reduced that have asize suitable for the amplification (e.g. larger than 70 bp), since manyfragments are cut to such a small size that they are for instanceselected out by the purification steps and are no longer in the sizewindow of the amplifiable nucleic acids. On the other hand, the numberof those fragments is increased, which have a size suitable for theamplification (that is, are not too large for an efficient PCRamplification); that is, those fragments are reduced, which due to theirlarge size are not amplifiable anymore. Thereby, the number ofpotentially interesting and amplifiable fragments again increases.

Despite the increase in the number of non-methylation-specific cuts, onthe one hand the complexity related to the number of the fragments inthe mixture to be amplified is reduced, and on the other hand anincrease of the number of potentially interesting fragments (i.e. thosepossibly containing CpG islands or containing fragments having a higherdensity of CG dinucleotides compared to the average in the genome) isachieved. Compared to the use of only one non-methylation-specificrestriction enzyme, a reduction of the complexity of the mixture (numberof the nucleic acids in the mixture) to 1/10 is obtained; that is, by afactor 10 better compared to use of only one non-methylation-specificrestriction enzyme.

This is an essential and substantial advantage over prior art methods,because a high complexity (i.e. amount of different nucleic acids orfragments) in the solution, to be tested for the presence of specificnucleic acids, will lead to unstable signals, increased crosshybridization and increased occurrence of non-specific hybridization.

Therefore, this embodiment of the method for preparing fragments inparticularly preferred in conjunction with the use of oligonucleotidearrays.

Another essential advantage, which is caused by the use of severalnon-methylation-specific restriction enzymes, is that generally fewervery long fragments are kept (remain) for the subsequent step withmethylation-specific restriction enzymes. Where the object is toidentify those fragments that are methylated over longer regions tocover several CG dinucleotides (so-called co-methylated regions, theyare of particular interest for the regulation of expression), then thisis not possible, if there is even a single methylation-specificrestriction cutting site in an unmethylated condition in this fragment.In this case, the fragment is cut into pieces and cannot be amplifiedanymore in the subsequent steps. Since it is now known that theso-called ‘co-methylation’ is often not 100%, and individualunmethylated CG cutting sites regularly exist, it is advantageous topreliminarily reduce the size of the fragments.

It is preferred that in step c) three different non-methylation-specificrestriction enzymes are added.

It is further preferred that at least one, preferably allnon-methylation-specific restriction enzymes cut recognition sequenceshaving a length of four bases, in particular recognition sequences,which do not contain CG. By using restriction enzymes with recognitionsequences having a length of four bases, the generation of fragmentsbeing long and thus disadvantageous for the amplification is preventedor reduced. Advantageously, at least one, preferably allnon-methylation-specific restriction enzymes generate sticky ends, inparticular sticky ends with an overhang containing TA. Particularlypreferred is the use of non-methylation-specific restriction enzymes,which cut a recognition sequence of four bases to sticky ends, and allrestriction enzymes produce the same overhangs. Alternatively, one orseveral non-methylation-specific restriction enzymes that produce stickyends can be used in conjunction with one or severalnon-methylation-specific restriction enzymes that produce blunt ends,because a ligation of a fragment with a sticky end with a fragment witha blunt end is also possible. Alternatively, It is also possible to useonly non-methylation-specific restriction enzymes that produce bluntends, since in this case a ligation is also possible. Thenon-methylation-specific restriction enzymes are preferably selectedfrom at least two, better three elements of the group consisting of“MseI, BfaI, Csp6I, Tru1I, Tvu1I, Tru9I, Tvu9I, MaeI and XspI”.Particularly preferred is the use of a combination of MseI, Bfa1 andCsp6. In principle, the step c) may be performed with common, (i.e.,simultaneous) addition of all non-methylation-specific restrictionenzymes to the solution. Alternatively, the restriction enzymes can besequentially added during the step c). In principle, everymethylation-specific restriction enzyme can be used. Preferably, themethylation-specific restriction enzyme is selected from the groupconsisting of “BisI, BstUI, BshI236I, AccII, BstFNI, McrBC, GlaI, MvnI,HpaII (HapII), HhaI, AciI, SmaI, HinP1I, HpyCH4IV, and mixtures of twoor more of the above enzymes”. Preferred, is a mixture containing therestriction enzymes BstUI, HpaII, HpyCH4IV and HinP1I.

The method according to the invention will normally be used for apolynucleic acid that is a naturally occurring DNA. Preferably, agenomic DNA is used (e.g., a human genomic DNA).

Herein, the DNA microarray will typically carry a plurality of differentnucleic acids containing known methylation sites. These can for instancebe obtained from gene databases.

In detail, the following can be made. A first solution with fragments ofa polynucleic acid, which originates from a tissue sample with diseasedtissue, is prepared. A second solution with fragments of a polynucleicacid, which originates from a tissue sample of the same tissue(s) typeadjacent to the diseased tissue, however being healthy tissue, isprepared. The first solution and the second solution are simultaneouslyor successively contacted with the DNA microarray and then hybridized.Such immobilized nucleic acids, in particular oligonucleotides, areselected, to which exclusively the fragments of the first solution or ofthe second solution are hybridized or not hybridized. By such a selectednucleic acid, DNA fragments are identified, which comprise regulatoryand/or coding regions of one or more genes. Thus, the correspondingproteins, peptides or RNAs are derived.

IV. Combination of Oligochips and Further Enrichment Methods

Additional embodiments provide for use of oligochips in conjunction withother enrichment methods, for instance a method analogous to the “NotIrepresentation” method according to WO 02/086163 (incorporated byreference herein in its entirety) or the method of the MS AP-PCR(Methylation Sensitive Arbitrarily-primed Polymerase Chain Reaction;Gonzalgo et al., Cancer Res. 57:594-599, 1997). Furthermore, enrichmentmay be performed by methods, which use the selective binding ofsubstances to methylated DNA. The enrichment may occur by means ofproteins, which methylation-specifically bind to the DNA, these may beMeCP2, MBD1, MBD2, MBD4 and Kaiso, or any domain thereof ormethylation-specific antibodies, e.g. anti-5-methylcytosine anti-bodies.Further, a chromatin immunoprecipitation (ChIP) may be performed for theenrichment. However, even further substances may be used for theenrichment, for instance triplex-forming PNA or DNA oligomers. Thementioned methods will be considered in detail hereunder.

Consequently, this inventive embodiment for determining the methylationpattern of polynucleic acids is characterized by the following steps:

a) a solution comprising a mixture of fragments of the polynucleic acidis made from a solution comprising the polynucleic acid, e.g. a solutioncomprising genomic DNA, wherein the composition of the fragment mixturedepends on the methylation pattern of the polynucleic acid;

b) the fragments of step a) are coupled with a substance beingdetectable with an optionally physical detection method, whereinoptionally an amplification of the fragments may occur;

c) a solution comprising the fragments of step b) is contacted with aDNA microarray, which carries a plurality of different immobilizedoligonucleotides, under conditions, at which a hybridization offragments occurs with correlated immobilized nucleic acids under definedstringency,

wherein the immobilized nucleic acids are selected from nucleic acids,which are specific for fragments of a genome, preferably the humangenome, and are localized on the DNA microarray at differentrespectively assigned positions;

d) optionally, a washing step is performed;

e) such nucleic acids, to which fragments of the solution are hybridizedand/or to which fragments of the solution are not hybridized, aredetected using the detection method;

f) optionally, from the detected hybridizations and/ornon-hybridizations according to step e) the methylation pattern of thepolynucleic acid being the educt of step a) is derived.

Oligoarray.

The structure and preparation of the oligonucleotide arrays aredescribed in detail above. Explicit reference is made to thecorresponding applications.

The oligonucleotides preferably have a length of 15 to 80 bases, inparticular 20 to 30 bases.

In a preferred embodiment, oligochips are used in combination withfragment enrichment methods, which comprise a digestion withnon-methylation-specific restriction enzymes, or which comprise a firstdigestion with non-methylation-specific restriction enzymes and a seconddigestion with methylation-specific restriction enzymes (in detail seeabove).

The sequences of the oligonucleotides can in particular be defined bythe following steps:

a) the genome of an organism is tested for first partial sequences,which are limited by cutting sites of the used non-methylation-specificrestriction enzymes and have a length of 100 to 1,200 base pairs, andsaid first partial sequences are selected;

b) from the first partial sequences, those are excluded, which containmore than 50% repeats, preferably such partial sequences are excluded,which contain more than 20% repeats, whereby a group of second partialsequences is formed, and wherein the steps a) and b) can be performed inany order.

For the case that methylation-specific restriction enzymes are furtherused for preparing the fragments, preferably in another step c), theselected second partial sequences are tested for cutting sites of theused methylation-specific restriction enzymes, and as third partialsequences those second partial sequences are selected, which containsuch cutting sites, and wherein the steps a) to c) can be performed inany order.

In another preferred variant of execution, oligochips are used incombination with fragment enrichment methods, which only comprise adigestion with methylation-specific restriction enzymes. Herein, thesequences of the oligonucleotides may in particular be defined by thefollowing steps:

a) the genome of an organism is tested for first partial sequences,which are limited by cutting sites of the used methylation-specificrestriction enzymes and have a length of 100 to 1,200 base pairs, andsaid first partial sequences are selected;

b) such partial sequences are excluded from the first partial sequences,which comprise more than 50% repeats, preferably, such partial sequencesare excluded, which contain more than 20% repeats, whereby a group ofsecond partial sequences is formed, and wherein the steps a) and b) canbe performed in any order.

In another preferred variant of execution, oligochips are used incombination with fragment enrichment methods, wherein fragments areenriched by a digestion with a first restriction enzyme andsimultaneously comprise a cutting site for a second restriction enzyme.Herein, the sequences of the oligonucleotides may in particular bedefined by the following steps:

a) the genome of an organism is tested for first partial sequences,which are limited by cutting sites of one or several of the first usedrestriction enzymes and have a length of 100 to 1,200 base pairs, andsaid first partial sequences are selected;

b) such partial sequences are excluded from the first partial sequences,which comprise more than 50% repeats, preferably such partial sequencesare excluded, which contain more than 20% repeats, whereby a group ofsecond partial sequences is formed, and wherein the steps a) and b) canbe performed in any order;

c) the selected second partial sequences are tested for cutting sites ofthe restriction enzymes used secondly, and as third partial sequencesthose second partial sequences are selected, which contain such cuttingsites, and wherein the steps a) to c) can be performed in any order.

In principle, it is also possible to combine every oligochip witholigonucleotides defined by one of the three above methods with eachenrichment method.

The partial sequences obtainable by the three mentioned methods maycorrespond to the sequences of the fragments obtained in step a). In apreferred embodiment, they are characterized in that they contain atleast one CG position within a methylation-specific restriction cuttingsite. Arbitrarily or according to further defined criteria,oligonucleotide sequences are now selected, which hybridize to thesepartial sequences (or their counter strand) or are identical to them (inorder to then hybridize to the counter strand).

It is preferred that these oligonucleotides are intended for thesynthesis on the DNA microarray. The above steps can be performed bymeans of simple programs based on publicly accessible gene databases. Itis further preferred that several oligonucleotide sequences usedhybridize to a fragment to be detected. Herein it is preferred that 3 to30 oligonucleotides hybridize to a fragment. It is particularlypreferred that 5 to 25 oligonucleotides hybridize to a fragment, and itis most particularly preferred that 10 to 20 oligonucleotides hybridizeto a fragment.

Alternately, said oligo sequences overlap in part.

In a preferred embodiment, exclusively oligonucleotide sequences areused for preparing the microarray, which hybridize in identical defineddistances to the complementary DNA to be tested. In this way, aso-called “tiling array” is created, as described for instance inKapranov P, Cawley S E, Drenkow J, Bekiranov S, Strausberg R L, Fodor SP, Gingeras T R. Large-scale transcriptional activity in chromosomes 21and 22 (Science 296:916-9, 2002). Thereby it is possible, in contrast tothe detection with specific fragments, to analyze the complete region ofa very large partial sequence, and thus conclusions can be drawn withregard to the presence or absence of a comethylation.

In a preferred embodiment, among the possible oligonucleotide sequencesthose are selected, which have the smallest signal/noise ratio and/orthe smallest cross hybridization rate.

Labeling.

It is preferred that the detectable substance is a fluorescence dye, andwherein the detection method comprises a selective scanning for thefluorescence radiation transmitted by the fluorescence dye,one-dimensional or two-dimensional, depending on the arrangement of thedifferent immobilized nucleic acids on the chip. The fluorescence dyemay be selected from the group consisting of “Cy3 and Cy5”. A person ofordinary skill in the art will be familiar with many other suitablefluorescence dyes.

In another preferred embodiment, the detectable substance may howeveralso be a biotin, which in the detection method interacts with anothersubstance and is detected thereby (see e.g. “Gene Chip Mapping AssayManual” of Affymetrix Inc.). Fragments from different samples areseparately hybridized on the microarrays, because they cannot bedistinguished by the detectable substance, thus two identical arrays areneeded, which have then to be standardized for the comparativeevaluation.

In a preferred embodiment of the invention, labeling is performed withthe detectable substance by amplification of the fragments. According toparticular aspects, so-called whole genome amplification methods areused (WGA—whole genome amplification, survey in: Hawkins et al.: Wholegenome amplification—applications and advances. Curr Opin Biotechnol.2002 Feb.; 13(1):65-7). In these methods, the fragments are amplified bymeans of a DNA polymerase and primers. The primers may belinker-specific primers, random primers or degenerated primers.

Up to now, different WGA methods are described. In the so-called primerextension preamplification (PEP), the amplification is performed bymeans of a random mixture of oligonucleotide primers having a length ofapprox. 15 nucleotides (Zhang et al.: Whole genome amplification from asingle cell: implications for genetic analysis. Proc Natl Acad Sci USA89:5847-51, 1992). In the DOP-PCR (degenerate oligonucleotide primedpolymerase chain reaction), however, only a degenerate primer is used(cf: Telenius et al.: Degenerate oligonucleotide-primed PCR: generalamplification of target DNA by a single degenerate primer; Genomics 13:718-25, 1992). Another WGA method is the so-called linker/adaptor-PCR.Therein, linkers are ligated to fragments. In the subsequentamplification, primers are used, which specifically bind to the linkers(survey in: Cheung and Nelson: Whole genome amplification using adegenerate oligonucleotide primer allows hundreds of genotypes to beperformed on less than one nanogram of genomic DNA. Proc Natl Acad SciUSA. 93:14676-9, 1996). The above WGA methods based on PCR have severaldrawbacks, however. For instance a generation of unspecificamplification artifacts may occur. Further, often an incomplete coverageonly of all genome regions will take place. Further, in part short DNAfragments with lengths of less than 1 kB only are generated. (cf: Deanet al.: Comprehensive human genome amplification using multipledisplacement amplification. Proc Natl Acad Sci USA. 99:5261-6, 2002).The most powerful method for a whole genome amplification is thereforeat present the isothermal “Multiple Displacement Amplification” (MDA,cf: Dean et al. 2002 as above; U.S. Pat. No. 6,124,120). The DNA isreacted with random primers and a DNA polymerase. Polymerases are usedhere, which are capable to displace the non-template strand of the DNAdouble strand during the amplification (e.g. a φ29 polymerase). Thedisplaced strands in turn serve as a matrix for the extension of furtherprimers. By using this method, an amplification by more than 5,000 ispossible. The average product length is more than 10 kB, and theamplification is distributed rather uniformly over the complete pool offragments. Commercial kits for the MDA are at present available from twosuppliers (“GenomiPhi” from Amersham Biosciences,www4.amershambiosciences.com; “Repli-g” from Molecular Staging,www.molecularstaging.com).

Execution of the Array.

The fragments detected with the immobilized oligonucleotides preferablycomprise nucleic acid sequences, the methylation sites of which are notmethylated or are methylated compared to the normal state, if a defineddisease exists. For this purpose, it not necessary that theoligonucleotide itself comprises this methylation site. The use of sucholigonucleotides, which comprise said methylation site, is one possibleexample of execution of the method.

The DNA microarray may exclusively carry oligonucleotides, which detectnucleic acid sequences (by hybridization), which are not methylated orare methylated compared to the normal state, if a defined diseaseexists. It is however also imaginable that on a microarray differentsets of oligonucleotides are immobilized, which cannot only detectdifferent fragments, which are specifically methylated for a disease,but also different sets of fragments, which in turn are specificallymethylated for different diseases or other conditions of interest, andthus the existence of a plurality of diseases or other conditions ofinterest characterized by a differential methylation can simultaneouslybe determined.

Other conditions of interest are for instance the risk to suffer from acertain disease, the prognosis of a certain type of a disease or thesusceptibility to side effects of a certain type of treatment. Alsodetectable are statements/determinations about the type or theaggressiveness or progress of a disease, for instance of a tumordisease, or about the efficiency of a therapy, if thesestatements/determinations are based on methylation differences.

Further it is possible to simultaneously perform SNP analyses on thesame microarray by means of another oligo set, and thus to generateeither further information about conditions based on genetic differences(SNP differentiation) or about the type or the aggressiveness orprogress of a disease, for instance a tumor disease, or about theefficiency of a therapy, if these statements are based on SNPdifferences.

The invention moreover relates to a method for preparing such arrayssuitable for the methylation analysis, which is characterized by thatthe oligonucleotides immobilized on the surface of the array are subjectto a selection, which is based on the method described above.

Test Kits.

The invention further relates to a test kit for performing a methodaccording to the invention, comprising a container and a DNA microarraycomponent, which carries a plurality of different immobilizedoligonucleotides at respectively assigned different places on the DNAmicroarray, and the oligonucleotide may contain at least one methylationsite. The oligonucleotides are further characterized by that they arenot longer than 200 bp.

Additional components of the test kit may be one or several of thefollowing components:

a restriction enzyme component or several different restriction enzymecomponents, the restriction enzymes of which are suitable for preparingthe fragments;

preferably a single restriction enzyme component, which comprisesexclusively one methylation-specific restriction enzyme or several ofsuch enzymes, preferably of a first restriction enzyme component, whichcomprises at least two different non-methylation-specific restrictionenzymes;

a second restriction enzyme component, which comprises exclusively onemethylation-specific restriction enzyme or several of such enzymes;

a protein component, the effective component of which binds DNAmethylation-specifically; and/or

a triplex-forming component, the effective component of whichdistinguishes between methylated and non-methylated DNA.

Another test kit additionally comprises a methylation-specificrestriction enzyme, wherein the oligonucleotides on the chip arecharacterized in that they hybridize to fragments, which comprise arestriction cutting site of at least one of the used methylationrestriction enzymes.

The oligonucleotides on the array can specifically hybridize to fragmentsequences, which comprise nucleic acid sequences, which are notmethylated or are methylated compared to the normal state, if a singledefined disease exists. Thereby the test kit would be suitable for thediagnosis of a specific disease. The disease may be a specific cancerdisease.

One or several of the following components, which usually are employedfor a DNA enrichment, may in addition be comprised: a linker or severallinkers, if applicable in a suitable solution; substances or solutionsfor performing a ligation; substances or solutions for performing acolumn chromatography; substances or solutions for performing animmunoprecipitation; substances or solutions for performing a PCR; a dyeor several dyes, if applicable with a coupling reagent, if applicable ina solution; substances or solutions for performing a hybridization;and/or substances or solutions for performing a washing step.

The invention further relates to a test kit for performing a methodaccording to the invention, comprising a container and a DNA microarraycomponent, which carries a plurality of different immobilized nucleicacids at respectively assigned different places on the DNA microarray,wherein the nucleic acids may comprise at least one methylation site.

Additional components of the test kit may be one or several of thefollowing components:

one restriction enzyme component or several different restriction enzymecomponents, the restriction enzymes of which are suitable for preparingthe fragments;

preferably one single restriction enzyme component, which comprisesexclusively one methylation-specific restriction enzyme or several ofsuch enzymes, preferably of a first restriction enzyme component, whichcomprises at least two different non-methylation-specific restrictionenzymes;

a second restriction enzyme component, which comprises exclusively onemethylation-specific restriction enzyme or several of such enzymes;

a protein component, the effective component of which binds DNAmethylation-specifically; and/or

a triplex-forming component, the effective component of whichdistinguishes between methylated and non-methylated DNA.

In another test kit, in addition a methylation-specific restrictionenzyme is comprised, the nucleic acids on the chip are characterized bythat they hybridize to fragments, which comprise a restriction cuttingsite of at least one of the used methylation restriction enzymes.

The nucleic acids on the array can specifically hybridize to fragmentsequences, which comprise nucleic acid sequences, which are notmethylated or are methylated compared to the normal state, if a singledefined disease exists. Thereby the test kit would be suitable for thediagnosis of a specific disease. The disease may be a specific cancerdisease.

One or several of the following components, which usually are employedfor a DNA enrichment, may in addition be comprised: a linker or severallinkers, if applicable in a suitable solution; substances or solutionsfor performing a ligation; substances or solutions for performing acolumn chromatography; substances or solutions for performing animmunoprecipitation; substances or solutions for performing a PCR; a dyeor several dyes, if applicable with a coupling reagent, if applicable ina solution; substances or solutions for performing a hybridization;and/or substances or solutions for performing a washing step.

Preparation of the Fragments.

The preparation of the fragments of the polynucleic acid may beperformed in the most various ways. According to the invention, anenrichment of methylated or not methylated fragments is performed. Theenrichment may be made in various ways. Substantially, on the one hand,an enrichment occurs methylation-specifically by targeted treatment ofthe DNA with restriction enzymes, and on the other hand, by bringing theDNA into contact with substances specifically binding methylated orunmethylated sequences.

Preparation of the Fragments by Restriction Enzyme Treatment.

According to the invention, several methods can be used for theenrichment by specific treatment of the DNA with restriction enzymes.Several methods have already been described above.

Method I):

In a preferred embodiment, the enrichment of methylated or unmethylatedfragments occurs by digestion of the DNA with at least onemethylation-specific restriction enzyme without previous addition of anon-methylation-specific restriction enzyme. For instance, the followingsteps may be provided:

a) a solution comprising the polynucleic acid is prepared;

b) optionally, a processing step is performed, in which substances thatare not polynucleic acids, are depleted, and/or the polynucleic acid isenriched;

c) a methylation-specific restriction enzyme or severalmethylation-specific restriction enzymes are added to the solutionwithout previous addition of a non-methylation-specific restrictionenzyme, wherein the polynucleic acid is cut to fragments at restrictionsites, which are capable of being methylated, but are not methylated;and

d) the fragments obtained in step c) are subjected to an amplificationstep, and fragments having a length in the range from 50 bases to 5,000bases are selectively enriched.

After the restriction, adapters are ligated to the fragments. Then anamplification of the fragmented DNA is performed, and simultaneously alabelling of the fragments by means of a detectable substance can beperformed.

Optionally, fragments after adapter ligation are subject to a digestionwith the same restriction enzyme or enzymes as used in step c) beforeamplification. This has the advantage that religated fragments aredigested while fragment-adapter ligations remain unaffected.

As methylation-specific restriction enzymes, enzymes may be used thatonly cut if their recognition sequence is unmethylated. A person ofordinary skill in the art is familiar with the respective restrictionenzymes. Examples for the used enzymes are: BstUI, BshI236I, AccII,BstFNI, MvnI, HpaII (HapII), HhaI, AciI, SmaI, HinP1I, HpyCH4IV, orcombinations of one or more of said enzymes are used. According toparticular aspects, such restriction enzymes may also be used that onlycut if a methylated recognition sequence exists. A person or ordinaryskill in the art will be familiar with the respective restrictionenzymes. Here, as examples only, McrBC (New England Biolabs) and therecently identified BisI (SibEnzyme Ltd.,www.science.sibenzyme.com/article8_article_(—)7_(—)1.phtml) and GlaI(SibEnyzme Ltd.,www.science.sibenzyme.com/article8_article_(—)11_(—)1.phtml) arementioned. The use of further enzymes not yet identified is imaginable,as far as they methylation-specifically cut, if methylated orunmethylated recognition sequences exist. Of course also a mixture ofsaid enzymes is applicable.

By this method, fragments having a length in the range from 50 bases to5,000 bases, preferably however from 50 to 2,000 bases, more preferablyfrom 80 to 2,000, most preferably from 100 to 2,000 bases, and inparticular from 100 to 1,000 bases are selectively enriched.

In the specified length window, thus a reduction of the complexity by afactor greater than 100 is achievable. This is also based on the factthat methylated sequence regions (or in another case unmethylatedsequence regions) and sequence regions without recognition sequences ofthe methylation-specific restriction enzymes used are not cut andconsequently form fragments, the length of which is regularly above theupper limits of the amplification window. In contrast thereto, regionswith unmethylated recognition sites (restriction enzyme, which cuts, ifan unmethylated recognition sequence exists) are cut and form fragmentshaving a length below the upper limit of the amplification window. Thesame will of course happen in the case of restriction enzymes, whichonly cut if their recognition sequences are methylated. Further, nopotentially interesting fragments are non-methylation-specifically cutand thereby reduced to a length below the lower limit of the window.Consequently, all interesting fragments, i.e. those with potentiallyhypermethylated or hypomethylated sites, are available for the followinganalyses. Finally, the full process is simplified, since lessrestriction enzymes are used. It is even possible to perform allreactions up to the hybridization on a DMH chip in one vessel (one tubeprocess). Consequently, processing is simplified and considerablyfaster. Finally, the number of potential error sources in the process issubstantially reduced.

Method II.

In a preferred embodiment, the enrichment of methylated or unmethylatedfragments occurs by digestion with non-methylation-specific restrictionenzymes and after ligation of adapters to the fragments, if applicablewith methylation-specific enzymes. For instance, the following steps maybe provided:

a) a solution comprising the polynucleic acid is prepared;

b) optionally, a processing step is performed, in which sustances thatare not polynucleic acids, are depleted, and/or the polynucleic acid isenriched;

c) one or preferably at least two different non-methylation-specificrestriction enzymes are added to the solution, wherein the polynucleicacid is cut at cutting sites being specific for the restriction enzymes;

d) the solution obtained in step c) is purified while separating smallfragments;

e) linkers are ligated to the fragments obtained in step d);

f) then one or preferably at least two methylation-specific restrictionenzymes are added to the solution obtained in step e), the fragmentsobtained in step d) being cut at cutting sites, which are capable ofbeing methylated, but are not methylated, or the fragments obtained instep d) being cut at cutting sites, which are capable of beingmethylated and are actually methylated; and

g) the fragments obtained in step f) are subjected after a furtherpurification step performed as an option to an amplification step, thosefragments being amplified, which were not cut in step f).

In a preferred embodiment, in step d) of the above method, fragmentshaving a length of less than 40 bp, preferably less than 70 bp,particularly preferably less than 100 bp are separated from the solutionobtained in step c).

In a preferred embodiment, the amplification in step g) takes place bymeans of primer molecules, which hybridize to the linkers introduced instep e) and of a polymerase under suitable PCR conditions.

Preferably, thus fragments having a length in the range from 50 bases to5,000 bases, preferably however from 70 to 2,000 bases, and inparticular from 100 to 1,200 bases are selectively enriched.

Thereby, a solution with a mixture of fragments of a polynucleic acid isobtained, which is particularly suitable for the method according to theinvention. For this method, it is particularly preferred that in step c)at least two non-methylation-specific restriction enzymes are used. Inthe case of at least two non-methylation-specific restriction enzymes,on the one hand the number of those fragments is reduced, which have asize suitable for the amplification (e.g. larger than 70 bp), since manyfragments are cut to such a small size that they are for instanceselected out by the purification steps and are no longer in the sizewindow of the amplifiable nucleic acids. On the other hand, the numberof those fragments is increased, which have a size suitable for theamplification (that is, are not too large for an efficient PCRamplification), i.e. those fragments are reduced, which due to theirlarge size are not amplifiable anymore. Thereby, the number ofpotentially interesting and amplifiable fragments again increases.

Despite the increase in the number of non-methylation-specific cuts, onthe one hand the complexity related to the number of the fragments inthe mixture to be amplified is reduced, and on the other hand anincrease of the number of potentially interesting fragments (i.e.possibly containing CpG islands or containing fragments having a higherdensity of CG dinucleotides compared to the average in the genome) isachieved. Compared to the use of only one non-methylation-specificrestriction enzyme, a reduction of the complexity of the mixture (numberof the nucleic acids in the mixture) to 1/10 is obtained (i.e. by afactor 10 better than when using only one non-methylation-specificrestriction enzyme).

This is an essential advantage over prior art methods, since a highcomplexity (i.e. amount of different nucleic acids or fragments) in thesolution, which is to be tested for the presence of specific nucleicacids, will lead to unstable signals, increased cross hybridization andincreased occurrence of unspecific hybridization. Therefore, thisembodiment of the method for preparing the fragments is particularlypreferred in conjunction with the use of oligonucleotide arrays.

Another essential advantage, which is caused by the use of severalnon-methylation-specific restriction enzymes, is that generally fewervery long fragments are maintained for the step withmethylation-specific restriction enzymes. Where the object is toidentify those fragments, which are methylated over longer regions toseveral CG dinucleotides (so-called co-methylated regions, of particularinterest for the regulation of the expression), then this is notpossible if there is even a single methylation-specific restrictioncutting site in an unmethylated condition in this fragment. In thiscase, the fragment is cut into pieces and cannot be amplified anymore inthe following. Because it is now known that the so-called co-methylationis often not 100% and individual unmethylated CG cutting sites regularlyexist, it is advantageous to preliminarily reduce the size of thefragments.

Preferably, in step c), three different non-methylation-specificrestriction enzymes are added.

It is further preferred that at least one, preferably allnon-methylation-specific restriction enzymes cut recognition sequenceshaving a length of four bases, in particular recognition sequences,which do not contain CG. By using restriction enzymes with recognitionsequences having a length of four bases, the generation of fragmentsbeing short and thus separable by purification is increased, whichreduces the complexity. Simultaneously, by using restriction enzymeswith recognition sequences having a length of four bases, the number ofpotentially interesting fragments (i.e. fragments, which possiblycomprise CpG islands or fragments with a density of CG dinucleotideswith an amplifiable size being increased compared to the average in thegenome) is increased.

Advantageously, at least one, preferably all non-methylation-specificrestriction enzymes generate sticky ends, in particular sticky ends withan overhang containing TA. Particularly preferred is the use ofnon-methylation-specific restriction enzymes, which cut a recognitionsequence of four bases to sticky ends, and all restriction enzymesproduce the same overhangs. Alternately, one or severalnon-methylation-specific restriction enzymes that produce sticky endsare used in conjunction with one or several non-methylation-specificrestriction enzymes that produce blunt ends, since a ligation of afragment with a sticky end with a fragment with a blunt end is alsopossible. Of course it is also possible to use onlynon-methylation-specific restriction enzymes, which produce blunt ends,since in this case, too, a ligation is possible. Thenon-methylation-specific restriction enzymes are preferably selectedfrom at least two, better three from of the group consisting of MseI,BfaI, Csp6I, Tru1I, Tvu1I, Tru9I, Tvu9I, MaeI and XspI. Particularlypreferred is the use of a combination of MseI, Bfa1 and Csp6. Inprinciple, the step c) may be performed with common (i.e. simultaneous)addition of all non-methylation-specific restriction enzymes to thesolution. It is also possible to add the restriction enzymessequentially to the solution during the step c). In principle, everymethylation-specific restriction enzyme can be used. Preferably, themethylation-specific restriction enzyme is selected from the groupconsisting of BisI, BstUI, BshI236I, AccII, BstFNI, McrBC, GlaI, MvnI,HpaII (HapII), HhaI, AciI, SmaI, HinP1I, HpyCH4IV, and mixtures of twoor more of the above enzymes. Preferred is a mixture containing therestriction enzymes BstUI, HpaII, HpyCH4IV and HinP1I.

Method III):

In a preferred embodiment, the enrichment of the unmethylated fragmentsmay substantially occur by means of the method of the “NotIrepresentation” method according to WO 02/086163 (incorporated byreference herein in its entirety). In the “NotI representation” method,genomic DNA is digested with suitable restriction enzymes or with BamHIand BgIII. After inactivation of the enzymes, the fragments arecircularized by self-ligation. The circularized DNA is then subjected toanother digestion with the methylation-specific restriction enzyme NotI.This enzyme cuts the DNA only if its recognition sequence isunmethylated. Therefore, the majority of all circularized fragments isnot digested and continues being circularized, since the fragmentseither do not contain a NotI cutting site, or they contain a methylatedNotI cutting site. Only fragments with an unmethylated NotI cutting siteare linearized by this step. To said fragments linearized again,NotI-specific linkers (adapters) are ligated, by means of which thefragments can be amplified in a subsequent PCR. In this way it ispossible to enrich fragments, which have unmethylated cutting sites.

As mentioned above (prior art), CpG dinucleotides are concentrated inCpG islands. a person skilled in the art is familiar with that normallyall CpG dinucleotides within a CpG island have the same methylationstate (co-methylation), i.e. they are either all methylated or allunmethylated (for comparison see Eads C A, Danenberg K D, Kawakami K,Saltz L B, Blake C, Shibata D, Danenberg P V, Laird P W. MethyLight: ahigh-throughput assay to measure DNA methylation. Nucleic Acids Res.2000 Apr. 15; 28(8); or Raykan V K, Hildmann T, Novik K L, Lewin J, TostJ, Cox A V, Andrews T D, Howe K L, Otto T, Olek A, Fischer J, Gut I G,Berlin K, Beck S. DNA methylation profiling of the human majorhistocompatibility complex: a pilot study for the human epigenomeproject. PLoS Biol. 2004 December; 2(12)). By the enrichment offragments with unmethylated NotI cutting sites, thus unmethylatedfragments are enriched.

Method IV:

In a preferred embodiment, the enrichment of the unmethylated fragmentsmay essentially occur according to the MS AP-PCR (Methylation SensitiveArbitrarily-primed Polymerase Chain Reaction). a person of ordinaryskill in the art will be familiar with this method, which was initiallydescribed by Gonzalgo et al., Cancer Res. 57:594-599, 1997. In thismethod, genomic DNA is digested with one or several restriction enzymes,for instance HpaII. The fragments thus obtained are used in a PCRamplification, and the used primers arbitrarily bind to the DNA (randomprimers) and further are CG-rich. By using such arbitrary CG-richprimers, preferably DNA sections are amplified that contain CGdinucleotides.

Enrichment of DNA by Means of Substances, which Bind to Methylated DNA.

In a preferred variant, the enrichment of the methylated or notmethylated fragments may occur by using substances, which selectivelybind to methylated or not methylated DNA. The binding may take place ina sequence-specific manner as well as in a sequence-unspecific manner.After binding to the substances, the bound DNA can be separated from theunbound DNA. Depending on whether the DNA to be detected is methylatedor unmethylated, the bound or the unbound fraction can further beanalyzed.

For the method according to the invention, different substancesmethylation-specifically binding to the DNA may be used.

Enrichment of DNA by Means of Proteins:

In a preferred embodiment, the enrichment occurs by means of proteins ortheir domains methylation-specifically binding to the DNA. Several suchproteins are known, inter alia MeCP2, MBD1, MBD2, MBD4 and Kaiso (surveyin: Shiraishi et al. Methyl-CpG binding domain column chromatography asa tool for the analysis of genomic DNA methylation. Anal Biochem.329:1-10, 2004; incorporated by reference herein in its entirety;Henrich et Tweedie: The methyl-CpG binding domain and the evolving roleof DNA methylation in animals. Trends Genet. 19:269-77, 2003; bothincorporated by reference herein in its entirety).

By means of proteins methylation-specifically binding to the DNA, amethylation-specific enrichment may occur in various ways. It is forinstance possible to use proteins specifically binding methylated DNA aswell as proteins specifically binding unmethylated DNA. Further, it ispossible to bind that DNA that is to be detected later. For thispurpose, first the unbound DNA is separated, and then the bound DNA isremoved from the protein. It is also possible to bind the background DNAto the protein and to remove it then from the reaction batch. As well, acombination of proteins is possible, wherein a protein specificallybinds methylated DNA, and another protein specifically bindsunmethylated DNA. This has the advantage that simultaneouslyunmethylated DNA and methylated DNA are enriched, whereas DNA with no orfew CpG positions are separated.

The protein binding and the separation of the bound and unbound DNA maytake place in various ways. It is for instance possible to bind theproteins to a solid surface, for instance in the form of beads for theseparation in the batch method or in the form of a column (cf: Cross etal., Nature Genetics 6:236-244, 1994). The unbound DNA can then beremoved by washing steps. Further it is possible to have the binding tothe proteins take place in the solution, and then to separate the DNAprotein complexes by usual methods such as centrifugation orchromatography from the unbound DNA. a person skilled in the art isfamiliar with biochemical methods to be used, for instance by usingbiotinylated proteins or proteins provided with Histidine-tag (forinstance Gretch et al. Anal Biochem 163:270-7, 1987; Janknecht et al.Proc. Natl Acad Sci USA 88:8972-6, 1991).

In a particularly preferred embodiment, the enrichment occurs by theso-called MDB column chromatography, which is described in detail byShiraishi et al., 2004, ibid. Explicit reference is made to thispublication.

The methyl-CpG-binding domain of the MeCP2 protein can be used for thispurpose, said domain specifically binding methylated, not howeverunmethylated or hemimethylated cytosines. The corresponding domainexpressing in vitro may for instance be bound to a modified agarosesurface by additional histidine residues. The domain detectssequence-unspecifically methylated CpG positions. The binding of themethylated DNA to the column occurs in dependence on the methylationdegree and the density of the CpG positions. The bound methylated DNAmolecules can then be eluted by increasing the salt concentration andsubsequently analyzed (see in detail: Shiraishi et al. 2004, ibid.).Besides that, it is also possible to analyze the unbound, unmethylatedfraction.

Besides that, it is also possible to enrich methylated and unmethylatedDNA by means of the CXXC-3 domain of the MBD1 protein. This domain cansequence-unspecifically bind to unmethylated CpG positions (Jørgensen,H. F., Ben-Porath, I., Bird, A. P. Molecular and Cellular Biology,3387-3395, 2004). The corresponding domain expressed in vitro may forinstance be bound by additional histidine residues to a modified agarosesurface. The binding of the unmethylated DNA to the column occurs independence on the methylation degree and the density of the CpGpositions. The bound unmethylated DNA molecules can then be eluted byincreasing the salt concentration and subsequently analyzed.

Additionally, it is also possible to analyze the unbound, methylatedfraction.

In a particularly preferred embodiment, the enrichment occurs by usingseveral different proteins or protein domains in combination. Inparticular it is preferred to first enrich unmethylated DNA by means ofa column, to which the CXXC-3 domain of the MBD1 protein is coupled. Thenon binding DNA may then be used to enrich methylated DNA. In this way,it is possible to simultaneously enrich unmethylated DNA and methylatedDNA, and further DNA is separated, which has no or only few CpGpositions.

Additionally, it is possible first to enrich methylated DNA andthereafter unmethylated DNA. In this case, too, DNA is separated, whichhas no or only few CpG positions.

According to the principle named above, other proteins or proteindomains methylation-specifically binding to DNA may also be used for theenrichment, in particular proteins sequence-specifically binding to CpGpositions, for instance by means of the kaiso protein, which detectssymmetrically methylated CpGpCpG positions.

Besides the MDB proteins mentioned above, in principle further proteinsmethylation-specifically detecting DNA may also be used. Thereto belongfor instance restriction enzymes or methyltransferases. It is imaginablethat those parts of said enzymes, which are responsible for themethylation-specific binding, are used for an enrichment without thecorresponding active center.

Enrichment of DNA by Means of Antibodies:

In another preferred embodiment, the enrichment occurs bymethylation-specific antibodies. Anti-5-methylcytosine antibodies areknown and commercially available since long times (www.abcam.com; AbcamInc; One Kendall Square; Bldg. 200, 3^(rd) Floor; Cambridge, Mass.02139). These antibodies may also be bound to a column or be bound in asolution to the DNA by the known methods. Details thereof are known to aperson skilled in the art (for instance Fisher et al. Nucleic Acids Res.32:287-97, 2004).

Moreover, an immunoprecipitation may be performed withanti-5-methylcytosine antibodies. The DNA antibody complexes areprecipitated in a suitable way, for instance with correspondingsecondary antibodies. The fragments thus enriched are released from theproteins, for instance, by proteinase K digestion.

In one embodiment, the DNA may be preliminarily randomly or not randomlyfragmented, and this can take place in an art-recognized manner. As arandom fragmentation method, the treatment with ultrasound or shearingis particularly preferred. As a non-random fragmentation method, afragmentation with methylation-specific restriction enzymes isparticularly preferred. In principle, every methylation-specificrestriction enzyme can be used. Preferably, the methylation-specificrestriction enzyme is selected from the group consisting of BisI, BstUI,BshI236I, AccII, BstFNI, McrBC, GlaI, MvnI, HpaII (HapII), HhaI, AciI,SmaI, HinP1I, HpyCH4IV and mixtures of two or more of said enzymes.

Enrichment of DNA by Chromatin Immunoprecipitation:

In a preferred embodiment, a chromatin immunoprecipitation (ChIP) isperformed for the enrichment. (Details of this method are known topersons skilled in the art and can for instance be found in: Matarazzoet al. In vivo analysis of DNA methylation patterns recognized byspecific proteins: coupling CHIP and bisulfite analysis. Biotechniques.37:666-8, 670, 672-3, 2004). An immunoprecipitation with antibodies isperformed, which are directed against 5-methylcytosine-binding proteins.Said proteins are the proteins mentioned above already: inter aliaMeCP2, MBD1, MBD2, MBD4 and Kaiso (survey in: Shiraishi et al., ibid.).

Essentially, this method is based on the fact that in the presence ofprotease inhibitors, a fixation of the proteins to the DNA takes place,for instance by formaldehyde. After an ultrasonic treatment, theimmunoprecipitation is performed with antibodies, which specificallydetect methylation-specific proteins. This may for instance be made withanti-MeCP2 antibodies (Santa Cruz Biotechnology, Santa Cruz, Calif.,USA). The DNA/protein complexes are then precipitated with protein Asepharose or suitable secondary antibodies. The separation of theprotein from the DNA occurs in a conventional way, e.g. by heating oradding proteinase K.

In a preferred variant, the DNA is purified and fragmented in a suitableway by restriction digestion. Thereafter, an incubation with the5-methylcytosine binding proteins is performed. Then theimmunoprecipitation takes place as described above.

In another preferred variant, the DNA/protein complexes are isolatedbefore the second precipitation step by suitable physical methods suchas ultracentrifugation. A respective kit is already commerciallyavailable (Panomics, Inc., Redwood City, Calif., USA), and may be usedfor the method according to the invention.

In one embodiment, the DNA may be randomly or not randomly fragmented.(e.g., in any suitable art-recognized manner). As a random fragmentationmethod, the treatment with ultrasound or shearing is particularlypreferred. As a non-random fragmentation method, a fragmentation withmethylation-specific restriction enzymes is particularly preferred. Inprinciple, every methylation-specific restriction enzyme may be used.Preferably, the methylation-specific restriction enzyme is selected fromthe group consisting of BisI, BstUI, BshI236I, AccII, BstFNI, McrBC,GlaI, MvnI, HpaII (HapII), HhaI, AciI, SmaI, HinP1I, HpyCH4IV andmixtures of two or more of said enzymes.

Enrichment of DNA by Means of Triplex-Forming Molecules:

Besides proteins, according to the invention, further substances mayalso be used that are capable of methylation-specific binding to the DNA(e.g, triplex-forming PNA or DNA oligomers). Corresponding oligomers aredescribed in detail, for example, in the patent applicationPCT/EP04/06534 (applicant: Epigenomics AG), and the literature isreplete with descriptions as to how triplex-binding molecules can beused for isolating methylated DNA.

In an additional embodiment, triplex formation is used for separatingmethylated DNA from unmethylated DNA. The DNA is brought into contactwith a triplex-forming molecule, whereupon the triplex-forming moleculepreferably generates a triplex with the unmethylated DNA rather thanwith the methylated DNA, which is used for separation. Particularlypreferred is the triple helix affinity chromatography (cf: Triplexes andbiotechnology. In: Malvy, C., Harel-Bellan, A., Pritchard, L. L., eds:Triple helix-forming oligonucleotides. Kluwer Academic Publishers 1999,285, 287f with further quotations).

In a further embodiment, the DNA may initially be randomly ornon-randomly fragmented, by a suitable art-recognized method. As arandom fragmentation method, the treatment with ultrasound or shearingis particularly preferred. As a non-random fragmentation method, afragmentation with methylation-specific restriction enzymes isparticularly preferred. In principle, every methylation-specificrestriction enzyme may be used. Preferably, the methylation-specificrestriction enzyme is selected from the group consisting of BisI, BstUI,BshI236I, AccII, BstFNI, McrBC, GlaI, MvnI, HpaII (HapII), HhaI, AciI,SmaI, HinP1I, HpyCH4IV and mixtures of two or more of said enzymes.

General:

The inventive methods will normally be used for a polynucleic acid thatis a naturally occurring DNA. Preferably, a genomic DNA is used (e.g., ahuman genomic DNA).

In a preferred embodiment, the polynucleic acid is derived fromparaffin-embedded tissue. In an alternate preferred embodiment, thepolynucleic acid is derived from formalin-fixed tissue. According toparticular aspects, tissues which were treated with other fixatives mayalso be used as a source of the polynucleic acid. In general, thepolynucleic acid of any tissue which was subject to any chemical orphysical treatment may be used according to the invention. In apreferred embodiment, the polynucleic acid is derived from afresh-frozen tissue.

In a particularly preferred embodiment, inventive results obtained withmethylated DNA are compared to inventive results obtained withunmethylated DNA. For this purpose, the methylated DNA and theunmethylated DNA are initially enriched using one or more of the methodsdescribed. If necessary, the DNA is labelled and amplified before themethylated DNA is brought into contact with the inventive array, and theunmethylated DNA is brought into contact with the same inventive array.Such contacting respectively occurs under conditions affording ahybridization of the methylated or unmethylated fragments withcorrelated immobilized nucleic acids under a defined stringency. Afteran optional washing step, the spatially resolved detection of suchnucleic acids, to which fragments of the solution are hybridized and/orto which fragments of the solution are not hybridized, takes place. Bycomparison of the detected hybridizations and/or the detectednon-hybridizations obtained for the originally methylated DNA with thedetected hybridizations and/or the detected non-hybridizations obtainedfor the originally unmethylated DNA, the methylation pattern of the DNAsused can be derived. This embodiment has the advantage of a very highsensitivity.

Further, the inventive method can also be used in combination with anarray, wherein the immobilized nucleic acids consist of more than 80bases. These nucleic acids may for instance be fragments containing atleast one CpG dinucleotide.

Therein, the DNA microarray will typically carry a plurality ofdifferent nucleic acids, which comprise known methylation sites. Theseare for instance obtainable from gene databases.

In detail, the following can be made. A first solution with fragments ofa polynucleic acid, which originates from a tissue sample with diseasedtissue, is prepared. A second solution with fragments of a polynucleicacid, which originates from a tissue sample of the same tissues typeadjacent to the diseased tissue, however with healthy tissue, isprepared. The first solution and the second solution are simultaneouslyor successively contacted with the DNA microarray and then hybridized.Such immobilized nucleic acids, in particular oligonucleotides, areselected, to which exclusively the fragments of the first solution or ofthe second solution are hybridized or not hybridized, therebyidentifying DNA fragments that comprise regulatory and/or coding regionsof one or several genes. Accordingly, the respective proteins, peptidesor RNAs are also derived.

Further it is possible to use the inventive oligonucleotide arrays foridentifying so-called expressed CpG islands sequence tags (ECIST). For adetailed description of this method, reference is made to U.S. Ser. No.60/118,760 and the quotations therein (incorporated by reference in itsentirety). According to the invention, the hybridization of twodifferent samples on the same DNA array is compared. The one sample wasgenerated from genomic DNA, and the other one originates from mRNA. Byhybridization with the sample originating from genomic DNA, regions ofthe genome that are subject to a differential methylation aredetermined. By means of the second sample, regions that are expressedcan be determined. Since in both cases, the same DNA chip is used, asimple comparison reveals which regions of the genome are subject tosuch a differential methylation and are simultaneously expressed in areciprocal differential manner.

V. Preprocessing of Signal Intensities

In particular preferred embodiments, as is familiar in the art, detectedsignal intensities are used directly for statistical analysis withoutany prior signal preprocessing. This is in particular the case: i) ifonly signal intensities which are derived from the same microarray-chipare compared with each other; ii) if controls distributed over themicroarray-chip have the same signal intensities; and iii) or both. Thecontrols are thereby characterized in that they have the same degree ofmethylation and they are distributed randomly, evenly or randomly andevenly over the entire microarray-chip.

In another preferred embodiment, different sets of controls are used,wherein each set is characterized in that: i) each control within a sethas the same degree of methylation as any other control of the same set;ii) the controls of different sets differ in their degree ofmethylation; and iii) the controls of a set are distributed randomly,evenly or randomly and evenly.

However, it may also be favourable in certain cases to ‘preprocess’detected signal intensities. Such cases can be one or a combination ofthe following situations: i) signal intensities are derived fromdifferent microarray-chips and are compared with each other; ii)controls, as specified above, have different signal intensities; or iii)preprocessing of signal intensities leads to more reliable andreproducible results compared to the use of signal intensities withoutpreprocessing.

According to a particular preferred embodiment, such preprocessingcomprises a “Log Transformation.” “Log Transformation” as used herein,stands for applying a logarithmic function for each signal intensityvalue, where the base can be any positive real number other than “1.” Ina preferred embodiment, the logarithm to the base X is applied for eachsignal intensity, whereby XεR⁺ other than “1”, preferably X is “1.3756”,“2”, “π”, “5”, “5.14”, “8.2754319”, “10”, “50.354”, or “10,000”. Thelogarithmic function is thereby of the following formula:log_(x)(signal intensity value)XεR⁺ other than 1, preferably X is 1.3756, 2, π, 5, 5.14, 8.2754319, 10,50.354, or 10,000.

In a preferred embodiment, a “Log-transformed” signal intensity value issubject to further preprocessing. In an alternate preferred embodiment,the signal intensity value was already preprocessed before a “LogTransformation” is applied to it. In a particular preferred embodiment,a “Log-transformed” signal intensity value is directly used forsubsequent analysis of the methylation status.

According to a preferred embodiment, the preprocessing comprises a“Quantile Normalization”. After this mathematical operation, allmicroarray-chips considered have the same distribution of signalintensities. Suitable methods are well known in the art, and include,but are not limited to Bolstad et al., Bioinformatics 2003 (incorporatedby reference herein in its entirety). In a particular embodiment,“Quantile Normalization” is carried out as exemplified in example 23.

Preferably, values obtained from “Quantile Normalization” of signalintensity values are subject to further preprocessing. Preferably, thesignal intensity values were already preprocessed before the “QuantileNormalization” is applied. Alternately, values obtained from the“Quantile Normalization” of signal intensity values are directly usedfor subsequent analysis of the methylation status.

Preferably, preprocessing of signal intensity values comprises a“Baseline Shift”. For this operation, the arithmetic mean value ofsignal intensity values of controls is subtracted from each signalintensity value of the considered microarray-chip. The controls arethereby characterized in one or a combination of the following: I) onlycontrols are considered which are located on the same microarray-chip,and subsequently the calculated arithmetic mean value is subtracted formeach signal intensity value of said microarray-chip; II) the controlsare distributed randomly, evenly or randomly and evenly over themicroarray chip; III) all considered controls comprise the same degreeof methylation; IV) all considered controls comprise the same amount ofDNA; and V) the ratio of control to non-control on a microarray-chip isat least 1/5,000, preferably this ratio is in the range of 1/1,000-1/1,more preferably this ratio is in the range of 1/250-1/5, and mostpreferably this ratio is in the range of 1/70-1/10, in particular it ispreferred that this ratio is 1/50.

In a preferred embodiment, a value obtained from the “Baseline Shift” ofa signal intensity value is subject to further preprocessing. In aparticular embodiment, the signal intensity value was alreadypreprocessed before the “Baseline Shift” is applied. In an alternateembodiment, a value obtained from the “Baseline Shift” of a signalintensity value is directly used for subsequent analysis of themethylation status.

According to a preferred embodiment, the preprocessing of signalintensity values comprises the generation of a representative value forthe signal intensity values of a set of immobilized nucleic acids. Saidset is characterized in that all immobilized nucleic acid of a set arelocated in the proximity of each other in the genome. Preferably, theimmobilized nucleic acids are parts of a CpG island array clone.Preferably, the immobilized nucleic acids are oligonucleotides asdescribed above (see description of oligonucleotide chips, in particularsection III and IV). The set of immobilized oligonucleotides ischaracterized in that the oligonucleotides of said set are comprised bythe same partial sequences or DNA fragments as they are obtainedaccording to oligonucleotide design or enrichment (see description ofoligonucleotide chips, in particular section III and IV; see section Iand II).

In a preferred embodiment, the representative value for the signalintensity values of a set of immobilized nucleic acids is generated byselecting the median value from the signal intensity values of thenucleic acids within a set to be analysed. According to anotherpreferred embodiment, the representative value is generated by anotherart-recognized mathematical function or operation. In a preferredembodiment, the representative value is generated by calculating thearithmetic mean value. In an alternate preferred embodiment, therepresentative value is generated by calculating the trimmed mean value.In a further preferred embodiment, the representative value is generatedby calculating the weighted mean value. In a yet another preferredembodiment, the representative value is generated by applying any linearor non-linear function or any linear or non-linear mathematicaloperation which is able to be generated from a plurality of value onerepresentative value(s).

In a particular preferred embodiment, a representative value for thesignal intensity values of a set of immobilized nucleic acids is subjectto further preprocessing. On one aspect, the signal intensity valueswere already preprocessed before a representative value for the signalintensity values of a set of immobilized nucleic acids is generated. Inan alternate preferred aspect, a representative value for the signalintensity values of a set of immobilized nucleic acids is directly usedfor subsequent analysis of the methylation status.

In a particular preferred embodiment, the signal intensity values arepreprocessed as described above according to the following order:

1. “Log-Transformation”

2. “Quantile Normalization”

3. “Baseline Shift”

4. Generation of a representative value for the signal intensity valuesof a set of immobilized nucleic acids.

In another preferred embodiment, the signal intensity values arepreprocessed as described above according to the following order:

1. “Log-Transformation”

2. “Baseline Shift”

3. Generation of a representative value for the signal intensity valuesof a set of immobilized nucleic acids.

In yet another preferred embodiment, the signal intensity values arepreprocessed as described above according to the following order:

1. “Log-Transformation”

2. “Quantile Normalization”

3. Generation of a representative value for the signal intensity valuesof a set of immobilized nucleic acids.

In a further preferred embodiment, the signal intensity values arepreprocessed as described above according to the following order:

1. “Log-Transformation”

2. Generation of a representative value for the signal intensity valuesof a set of immobilized nucleic acids.

According to a particular embodiment, a detected signal intensity ispreprocessed, wherein the preprocessing comprises one or a combinationof the following:

-   -   “Log-Transformation”;    -   “Quantile Normalization”;    -   “Baseline Shift”; and    -   Generation of a representative value for the signal intensity        values of a set of immobilized nucleic acids.

According to one embodiment, the preprocessing is carried out in thefollowing order:

a) “Log-Transformation”;

b) “Quantile Normalization”

c) “Baseline Shift”; and

d) Generation of a representative value for the signal intensity valuesof a set of immobilized nucleic acids.

According to an alternate embodiment, the preprocessing is carried outin the following order:

a) “Log-Transformation”;

b) “Baseline Shift”; and

c) Generation of a representative value for the signal intensity valuesof a set of immobilized nucleic acids.

According to an additional preferred embodiment, the preprocessing iscarried out in the following order:

a) “Log-Transformation”;

b) “Quantile Normalization”; and

c) Generation of a representative value for the signal intensity valuesof a set of immobilized nucleic acids.

According to yet another preferred embodiment, the preprocessing iscarried out in the following order:

a) “Log-Transformation”;

b) Generation of a representative value for the signal intensity valuesof a set of immobilized nucleic acids.

Yet further embodiments comprise preprocessing of signal intensityvalues that takes into account other controls like nucleic acidsencoding repeats or random sequences.

VI. Analysis of Copy-Number Changes

A preferred embodiment of the invention comprises:

-   -   deriving DNA from a test sample and/or a reference sample;    -   optionally, enriching DNA from the derived DNA, preferably        methylated and/or unmethylated DNA is enriched;    -   labelling enriched DNA of the test sample and/or the reference        sample identically or differentially with one or more physically        detectable substances;    -   hybridizing labeled DNA on one or more DNA arrays, preferably on        one or more arrays as described in one or more embodiments of        the invention;    -   optionally, performing a washing step; and    -   performing a spacially resolved detection of signal intensities        of those nucleic acids, to which fragments are hybridized and/or        to which fragments are not hybridized;    -   comparing of the detected signal intensities of hybridizations        and/or of non-hybridizations for DNA derived from a test sample        and for DNA derived from a reference sample, wherein the        percentage of methylation and/or the copy-number for said DNA is        deduced.

In a preferred embodiment, an analysis of copy-number changes is carriedout by means of comparative genomic hybridization (CGH) analysis.Therefore one or more oligonucleotide arrays according to the inventionand/or DNA enrichment methods according to the invention are used. Themethylation pattern and the copy-number of DNA in a genome are herebyanalysed simultaneously.

For CGH analysis, DNA fragments derived from genomic DNA isolated fromtest and reference samples are labeled and hybridized to aDNA-microarray, in particular an oligonucleotide microarray according tothe invention as described above (see section III and IV). Of coursemore than two genomes or samples can be compared simultaneously ifdistinguishable labels are used.

According to the invention, hybridization can be performed in differentways. In a particularly preferred embodiment, identical arrays are used,on each array only DNA fragments derived from genomic DNA of a singlesample are hybridized.

In another particularly preferred embodiment, each sample of DNAfragments obtained from genomic DNA of different samples is labeleddifferentially. Thus the differentially labeled DNA can be applied tothe same array. Moreover, it is preferred that only some of DNA fragmentsamples are labeled differentially and hybridized to the same array. DNAfragments derived from other samples and labeled with the same labelsare hybridized on a different array.

In a further particularly preferred embodiment, DNA fragments derivedfrom one or more test samples are hybridized each to different identicalor all to the same array. The data resulting from these hybridizationsis then compared with data obtained previously for DNA fragments of areference sample.

According to the invention, it is preferred that the complexity ofgenomic DNA is reduced. This is in particular preferred if anoligonucleotide array is used. The reduction of complexity has theadvantage that the signal-to-noise ratio is increased. Therefore the CGHanalysis is characterized by a high reliability and reproducibility alsoif only small amount of genomic DNA as starting material are available.Because of the same reason, the reduction of complexity also allows theuse of low complexity array elements if desired.

Oligonucleotide Array:

In a preferred embodiment one or more oligonucleotide arrays accordingto the invention are used for CGH analysis as described above (seesection III and IV). According to this embodiment, DNA fragments aregenerated according to standard CGH protocols as they are known to thoseskilled in the art. For example DNA fragments are made by cleavinggenomic DNA with a restriction endonuclease, ligating the cleavedproducts to template oligonucleotides, and then performing a polymerasechain reaction (PCR) amplification using complementary oligonucleotidesin which preferential DNA fragments smaller than 1 kb are amplified. Therestriction endonuclease can be for example DpnII or BgIII which willresult in a complexity reduction of 70% and 2.5%, respectively (Lucitoet al. Genome Research, 10: 1726-1736, 2000).

According to this embodiment, variation of the DNA copy-number but notalteration in the methylation pattern in a genome are detectable. Theintensity of the hybridization signal obtained for DNA fragments of testand reference samples at a given location is proportional only to therelative copy-number of those sequences in the test and the referencegenome. Typically the reference genome is regarded as normal. Increasesand decreases in the intensity of the hybridization signal relative tothat of the reference sample indicate only variations of the DNAcopy-number in the genome of the test sample.

The use of an oligonucleotide array according to the invention for CGHanalysis has the advantage that variations of the DNA copy-number areselectively analysed for genomic regions comprising genes or regulatoryregions. As well known in the art, the sites for methylation, the CpGdinucleotides, are mainly associated with genes or regions of regulatoryfunction. Secondly, the fragments of the oligonucleotide array hybridizespecifically to CpG dense DNA fragments contained in the complexityreduced representation of the genome. Because of that, this embodimentis of particular interest for high resolution gene-by-gene mapping ofcopy number changes, or for direct combination of data of copy-numberchanges with methylation changes. Furthermore, the use of anoligonucleotide array according to the invention for CGH analysis isadvantageous because the oligonucleotide array according to theinvention is characterized i) by a high resolution, and ii) by aincreased signal-to-noise ratio because no oligonucleotide comprisesmore than 50%, preferentially more then 20% repeats.

In a preferred embodiment, the one or more oligonucleotide arrays forCGH analysis are so called tiling arrays. As described above theoligonucleotides of such a tiling array are characterized in that theyhybridize exclusively in constant to each other defined distances on thecomplementary DNA for analysis. The advantage of the use of such tilingarrays for CGH analysis is that it is possible to determine directly theapproximate length of the region with variations in copy-number and thatthe whole genome of interest can be analysed. In another preferredembodiment, the one or more oligonucleotide arrays used for CGH analysisare oligonucleotide arrays according to the invention and they areso-called tiling arrays. This means there are only oligonucleotides ableto hybridize exclusively in constant to each other defined distances onthe fragments of the CpG-islands, specifically to CpG dense DNAfragments contained in the complexity reduced representation of thegenome. The use of such tilling arrays for CGH analysis has theadvantage that it is possible to determine directly the approximatelength of the region with variations in copy-number and that the onlyregions of the genome of interest are analysed which are either genes orcorrespond to them.

Complexity Reduction by Enrichment of Methylated and/or UnmethylatedDNA:

In a preferred embodiment, CGH analysis is performed by using knownarrays and complexity reduced DNA, wherein the complexity reduction isachieved by enrichment of methylated or unmethylated DNA. The Array canbe any array suitable for CGH analysis. In particular this array is anoligonucleotide array or a DNA array carrying any type of nucleic acid(DNA, RNA or PNA) with various lengths. The enrichment of methylated orunmethylated DNA can be done as described above by restrictions enzymes,by bringing the DNA into contact with substances specifically bindingmethylated or unmethylated DNA, or by combinations thereof (see sectionI, II, III and IV).

According to this embodiment, the corresponding hybridization signalsrepresent a mixture of copy-number changes and methylation changes. Itis not possible to interpret the signal intensities in a way thatenables a deduction of the copy-number changes and/or the methylationchanges. Therefore, in another embodiment, it is preferred that the DNAderived from a sample is completely methylated or unmethylated. Suitablemethods are known to those skilled in the art. A treatment to generatecompletely methylated DNA can be any kind of treatment, preferably atreatment with a methyltransferase, in particular a treatment with themethyltransferase SssI. The treatment can take place before or inbetween the enrichment where appropriate. A treatment to generatecompletely unmethylated DNA can be any kind of chemical or enzymatictreatment, in particular the genomic DNA is subject to amplification aswell known to those skilled in the art. Such an amplification ispreferably carried out according to whole genome amplification methodsas described above (see section IV “Labeling”). Most preferably, theisothermal “Multiple Displacement Amplification” (MDA) is used. The DNAis reacted with random primers and a DNA polymerase. The polymerase iscapable to displace the non-template strand of the DNA double strandduring the amplification (e.g. a φ29 polymerase). The displaced strandsserve as a matrix for the extension of further primers.

According to this embodiment, variation of the DNA copy-number, but notalteration in the methylation pattern in a genome is detectable. Theintensity of the hybridization signal obtained for DNA fragments of testand reference samples at a given location is proportional only to therelative copy-number of those sequences in the test and the referencegenome. Typically the reference sample is regarded as normal. Increasesand decreases in the intensity of the hybridization signal relative tothat of the reference sample indicate only variations of the DNAcopy-number in the genome of the test sample.

Furthermore, this embodiment has also the advantage that variations ofthe DNA copy-number are selectively analysed for genomic regionscomprising genes and regulatory regions. The reason for this is that thecomplexity of the DNA is reduced by enrichment of unmethylated ormethylated DNA or by restriction of one or more non-methylation-specificrestriction enzyme and subsequent linker mediated amplification asdescribed below in detail. As is well known in the art, CpGdinucleotides (the site for cytosine methylation) are mainly associatedwith genes or regulatory regions. Therefore, this embodiment is also ofparticular interest for high resolution gene-by-ene mapping ofcopy-number changes, or for direct combination of data of copy-numberchanges with methylation changes.

According to particular aspects of the invention, the enrichment can becarried out as described as follows:

Method I:

In a particularly preferred embodiment, unmethylated or methylated DNAis enriched by restriction enzyme treatment according to method I. Inbrief, the enrichment occurs by digestion of the DNA with at least onemethylation-specific restriction enzyme without previous addition of anon-methylation-specific restriction enzyme. For example, the followingsteps are provided:

a) a solution comprising the polynucleic acid is prepared;

b) optionally, a processing step is performed, in which substances thatare not polynucleic acids, are depleted, and/or the polynucleic acid isenriched/accumulated;

c) a methylation-specific restriction enzyme or severalmethylation-specific restriction enzymes are added to the solutionwithout previous addition of a non-methylation-specific restrictionenzyme, wherein the polynucleic acid is cut to fragments at restrictionsites, which are capable of being methylated, but are not methylated,and d) the fragments obtained in step c) are subjected to anamplification step.

In preferred embodiments, the amplification is carried out by ligationof adapters to the fragments after the restriction. Thereafter, thefragmented DNA is amplified, wherein simultaneous labeling of thefragments with a detectable substance can be achieved.

Optionally, before amplification, fragments after adapter ligation aresubject to a digestion with the same restriction enzyme or enzymes asused in step c). This has the advantage that any religated fragments aredigested while fragment-adapter ligations remain unaffected.

As methylation-specific restriction enzyme, any enzymes may be used thatonly cut if their recognition sequence is unmethylated. A person ofordinary skill in the art is will have knowledge of suitable restrictionenzymes. Examples of such enzymes are: BstUI, BshI236I, AccII, BstFNI,MvnI, HpaII (HapII), HhaI, AciI, SmaI, HinP1I, HpyCH4IV or mixtures ofsaid enzymes. According to the invention, restriction enzymes may alsobe used that only cut if a methylated recognition sequence exists. Aperson of ordinary skill in the art will know of such suitablerestriction enzymes (e.g., McrBC enzyme (New England Biolabs) and therecently identified BisI enzyme (SibEnzyme Ltd., Russia,www.science.sibenzyme.com/article8_article_(—)7_(—)1.phtml) and the GlaIenzyme (SibEnzyme Ltd., Russia,www.science.sibenzyme.com/article8_article_(—)11_(—)1.phtml) arementioned). Additionally, combinations of said enzymes are applicable.The use of further enzymes not yet identified is within the scope of thepresent invention, insofar as the enzymes cut methylation-specificallymethylated or unmethylated recognition sequences.

According to this embodiment, fragments having a length in the rangefrom 50 bases to 5,000 bases, preferably from 50 to 2,000 bases, mostpreferably from 50 to 1,000 bases, and in particular from 50 to 600bases are selectively enriched.

Accordingly, a reduction of the complexity by a factor greater than 100is achievable in the specified length window. This is also based on thefact that methylated sequence regions (or in the alternative case,unmethylated sequence regions) and sequence regions without recognitionsequences of the used methylation-specific restriction enzymes are notcut and consequently form fragments, the length of which is as a ruleabove the upper limits of the amplification window. In contrast thereto,regions with unmethylated recognition sites (restriction enzyme whichcuts only an unmethylated recognition sequence) are cut and formfragments having a length below the upper limit of the amplificationwindow. Of course, the equivalent will happen in the case of restrictionenzymes, which cut only, if their recognition sequences are methylated.Further, and significantly, no potentially interesting fragments are cutnon-methylation-specifically, and thereby are reduced to a length belowthe lower limit of the window. Consequently, all interesting fragments,i.e. those with potentially hypermethylated or hypomethylated sites, areavailable for the following analyses. Finally, the full process issimplified, since fewer restriction enzymes are used. It is evenpossible to perform all reactions up to the hybridization on a array inone vessel (one tube process). Consequently, processing is simplifiedand considerably faster. Finally, the number of potential error sourcesin the process is substantially reduced.

Method II:

In a particularly preferred embodiment, unmethylated or methylated DNAis enriched by restriction enzyme treatment according to method II. Theenrichment of methylated or unmethylated fragments occurs by digestionwith non-methylation-specific restriction enzymes and after ligation ofadapters to the fragments, if applicable with methylation-specificenzymes. For instance, the following steps may be provided:

a) a solution comprising the polynucleic acid is prepared;

b) optionally, a processing step is performed, in which substances thatare not polynucleic acids, are depleted, and/or the polynucleic acid isenriched;

c) one or preferably at least two different non-methylation-specificrestriction enzymes are added to the solution, wherein the polynucleicacid is cut at cutting sites being specific for the restriction enzymes;

d) the solution obtained in step c) is purified while separating smallfragments;

e) linkers are ligated to the fragments obtained in step d);

f) then one or preferably at least two methylation-specific restrictionenzymes are added to the solution obtained in step e), the fragmentsobtained in step d) being cut at cutting sites, which are capable ofbeing methylated, but are not methylated, or the fragments obtained instep d) being cut at restriction sites, which are capable of beingmethylated and are actually methylated; and

g) the fragments obtained in step f) are subjected to an amplificationstep after an optional further purification step, wherein only thosefragments not cut in step f) are amplified.

In a preferred embodiment, in step d) of the above method, fragmentshaving a length of less than 40 bp, preferably less than 70 bp, and morepreferably less than 100 bp are separated from the solution obtained instep c).

In a preferred embodiment, the amplification in step g) takes place bymeans of primer molecules, which hybridize to the linkers introduced instep e) and of a polymerase under suitable PCR conditions.

Accordingly, preferably fragments having a length in the range of 50bases to 5,000 bases, preferably in the range of 70 to 2,000 bases, andin more preferably in the range of 100 to 1,200 bases are selectivelyenriched.

It is preferred that in step c) three different non-methylation-specificrestriction enzymes are added.

It is further preferred that at least one, preferably allnon-methylation-specific restriction enzymes cut recognition sequenceshaving a length of four bases, in particular recognition sequences,which do not contain CG. By using restriction enzymes with recognitionsequences having a length of four bases, the generation of fragmentsbeing short and thus separable by purification is increased, whichreduces the complexity. Simultaneously, by using restriction enzymeswith recognition sequences having a length of four bases, the number ofpotentially interesting fragments (i.e. fragments, which possiblycomprise CpG islands or fragments with a density of CG dinucleotideswith an amplifiable size being increased compared to the average in thegenome) is increased.

Advantageously, at least one, preferably all non-methylation-specificrestriction enzymes generate sticky ends, in particular sticky ends withan overhang containing TA. Particularly preferred is the use ofnon-methylation-specific restriction enzymes, which cut a recognitionsequence of four bases to sticky ends, and all restriction enzymesproduce the same overhangs. Alternately, one or severalnon-methylation-specific restriction enzymes that produce sticky endsare used in conjunction with one or several non-methylation-specificrestriction enzymes that produce blunt ends, since a ligation of afragment with a sticky end with a fragment with a blunt end is alsopossible. Of course it is also possible to use onlynon-methylation-specific restriction enzymes, which produce blunt ends,since in this case, too, a ligation is possible. Thenon-methylation-specific restriction enzymes are preferably selectedfrom at least two, preferably three from of the group consisting ofMseI, BfaI, Csp6I, Tru1I, Tvu1I, Tru9I, Tvu9I, MaeI and XspI.Particularly preferred is the use of a combination of MseI, Bfa1 andCsp6. In principle, step c) may be performed with common (i.e.simultaneous) addition of all non-methylation-specific restrictionenzymes to the solution. Alternatively, the restriction enzymes can beadded sequentially to the solution during step c). In principle, everymethylation-specific restriction enzyme can be used. Preferably, themethylation-specific restriction enzyme is an enzyme which cuts itsrecognition site only if it is unmethylated. Suitable enzymes are knownto those skilled in the art, also so far unknown suitable restrictionenzymes are useable. In a preferred embodiment the methylation-specificrestriction enzyme is selected from the group consisting of BisI, BstUI,BshI236I, AccII, BstFNI, McrBC, MvnI, HpaII (HapII), GlaI, HhaI, AciI,SmaI, HinP1I, HpyCH4IV, and mixtures of two or more of the aboveenzymes. Preferred is a mixture containing the restriction enzymesBstUI, HpaII, HpyCH4IV and HinP1I. In another preferred embodiment, themethylation-specific restriction enzyme is an enzyme which cuts itsrecognition site only if it is methylated. Suitable enzymes are known tothose skilled in the art, and other suitable enzyme will no doubt bediscovered or engineered in the future. In a preferred embodiment, themethylation-specific restriction enzyme is selected from the groupconsisting of BisI, McrBC, GlaI, and combinations of two or morethereof. A person a ordinary skill in the art will know how to adjustfollowing described embodiments for comparative genomic hybridisation.

Combination of CpG-Island Array and of Complexity Reduction byEnrichment of Methylated and/or Unmethylated DNA:

In a preferred embodiment, CGH analysis is performed by usingCpG-island-arrays in combination with complexity reduced DNA, whereinthe complexity reduction is achieved by enrichment of methylated orunmethylated DNA. The enrichment of methylated or unmethylated DNA canbe done as described above by restriction enzymes, by bringing the DNAinto contact with substances specifically binding methylated orunmethylated DNA, or by combinations thereof (see section I, II, III andIV).

This embodiment has the advantage that changes in the methylationpattern and variations in the DNA copy-number can be simultaneouslydetected. According to the invention, the intensity of the hybridizationsignal obtained for DNA fragments of test and reference samples at agiven location is proportional not only to the state of methylation butalso to the relative copy-number of those sequences in the DNA of thetest and the reference sample. Typically the DNA of the reference sampleis regarded as normal. Increases and decreases in the intensity of thehybridization signal of the test sample relative to that of thereference sample indicate alteration of the methylation of the analyzedCpG positions and/or of the DNA copy-number in the genome of the testsample.

Moreover, this embodiment has the advantage that variations of the DNAcopy-number are selectively analysed for genomic regions comprisinggenes or regulatory regions. This is based on the following: First, thecomplexity of the DNA is reduced by enrichment of unmethylated ormethylated DNA or by restriction of one or more non-methylation-specificrestriction enzyme and subsequent linker mediated amplification asdescribed above in detail. As well known in the art, CpG dinucleotides(the site for cytosine methylation) are mainly associated with genes orregulatory regions. Second, the fragments of the CpG-island arrayhybridize specifically to CpG dense DNA fragments contained in thecomplexity reduced representation of the genome as produced by theembodiments of the invention. As is well known in the art, the sites formethylation (CpG dinucleotides) are primarily associated with genes orregulatory regions. Because of that, this embodiment is of interest forhigh resolution gene by gene mapping of copy number changes, or fordirect combination of data of copy-number changes with methylationchanges.

As described below, three major embodiments (embodiments I, II and III)are in particular preferred.

Combination of Oligonucleotide Array and of Complexity Reduction byEnrichment of Methylated and/or Unmethylated DNA:

In a particular preferred embodiment, CGH analysis is carried out bymeans of combining oligonucleotide arrays according to the invention(see section III and IV) and DNA enrichment methods according to theinvention (see section I, II, III and IV). This embodiment has theadvantage that changes in the methylation pattern and variations in theDNA copy-number can be simultaneously detected. According to thisaspect, the intensity of the hybridization signal obtained for DNAfragments of test and reference samples at a given location isproportional not only to the state of methylation but also to therelative copy-number of those sequences in the test and the referencegenome. Typically the reference genome is regarded as normal. Increasesand decreases in the intensity of the hybridization signal of the testsample relative to that of the reference sample indicate alteration ofthe methylation of the analyzed CpG positions and/or of the DNAcopy-number in the genome of the test sample.

According to the invention the following three major embodiments(embodiments I, II and III) are in particular preferred:

Embodiment I

A preferred embodiment comprises:

-   -   generating two types of samples of DNA fragments. each derived        from a test sample and/or a reference sample;    -   generating the first type of sample comprising a complexity        reduction of genomic DNA independent of the methylation pattern        of the genomic DNA;    -   generating the second type of sample comprising a first        methylation-non-specific restriction enzyme digestion and a        second methylation-specific restriction enzyme digestion;    -   deducing copy-number variations by comparison of detected signal        intensities of the first type of DNA fragment samples of a test        sample with the detected signal intensities of the first type of        DNA fragment samples of a reference sample; and    -   deducing methylation changes by comparison of detected signal        intensities of the second type of DNA fragment samples of a test        sample with the detected signal intensities of the second type        of DNA fragment samples of a reference sample.

Another preferred embodiment comprises a comparison of signalintensities derived from the first type of DNA fragment samples,wherein:

-   -   the same signal intensity for a test sample and for a reference        sample indicate that the genomic region in the test sample        corresponding to the hybridized DNA fragments is present in the        same copy-number as the corresponding genomic region in the        reference sample;    -   an increased signal intensity for a test sample in comparison to        the signal intensity for a reference sample indicates that the        genomic region in the test sample corresponding to the        hybridized DNA fragments is present at a higher copy-number than        the corresponding genomic region in the reference sample,        wherein the increase of the copy-number is thereby proportional        to the signal increase;    -   a decreased signal intensity for a test sample in comparison to        the signal intensity for a reference sample indicates that the        genomic region in the test sample corresponding to the        hybridized DNA fragments is present at a lower copy-number than        the corresponding genomic region in the reference sample,        wherein the decrease of the copy-number is thereby proportional        to the signal decrease.

A further preferred embodiment comprises a comparison of signalintensities derived from the second type of DNA fragment samples,wherein:

-   -   the same signal intensity for a test sample and for a reference        sample indicates that the same degree of analyzed cytosines of        the test sample is methylated as in the reference sample;    -   an increased signal intensity for a test sample in comparison to        the signal intensity for a reference sample indicates that a        higher degree of the analyzed cytosines in the test sample is        methylated than in the reference sample, wherein the increase in        methylation is thereby proportional to the signal increase; and    -   a decreased signal intensity for a test sample in comparison to        the signal intensity for a reference sample indicates that a        lower degree of the analyzed cytosines of the test sample is        methylated than in the reference sample, wherein the decrease in        methylation is thereby proportional to the signal decrease.

According to embodiment I, two types of samples of DNA fragments aregenerated each from genomic DNA of test and of reference samples. Thefirst type of sample (herein referred as type A) is generated byenrichment of DNA fragments independent of the methylation pattern ofthe genomic DNA. In a first embodiment, type-A DNA fragments aregenerated by enrichment according to the above-described method II,wherein genomic DNA is digested with non-methylation-specificrestriction enzymes, linkers are ligated to the resulting fragments, andfragments with linkers are amplified by linker mediated PCR.

According to a second embodiment, type-A DNA fragments are generated bytreatment of DNA so that all cytosines of CpG dinucleotides aremethylated and by enrichment of methylated DNA fragments. Accordingly,the enrichment is carried out according to the above-described methodII, wherein genomic DNA is digested with non-methylation-specificrestriction enzymes, linkers are ligated to the resulting fragments,linker-ligated fragments are digested with methylation-specificrestriction enzymes, and fragments with linkers are amplified by linkermediated PCR. The treatment which results in a complete methylation ofcytosines of CpG dinucleotides can be any kind of treatment, preferablya treatment with a methyltransferase, in particular a treatment with themethyltransferase SssI. The treatment can take place before or inbetween the enrichment, in particular before any digestion of genomicDNA with non-methylation-specific restriction enzymes, or before linkersare ligated to the resulting fragments, or before all fragments aresubjected to a methylation-specific restriction enzyme digestion.

According to a third embodiment, type-A DNA fragments are generated bytreatment of DNA so that all cytosines of CpG dinucleotides are notmethylated and by enrichment of unmethylated DNA fragment. Accordingly,the enrichment is carried out according to the above-described methodII, wherein genomic DNA is digested with non-methylation-specificrestriction enzymes, linkers are ligated to the resulting fragments,linker-ligated fragments are digested with methylation-specificrestriction enzymes, and fragments with linkers are amplified by linkermediated PCR. The treatment which results in a complete unmethylation ofcytosines of CpG dinucleotides can be any kind of chemical or enzymatictreatment, in particular the genomic DNA is subject to amplification aswell known to those skilled in the art. Such an amplification ispreferably carried out according to whole genome amplification methodsas described above (see section IV, “Labeling”). Most preferably theisothermal “Multiple Displacement Amplification” (MDA) is used. The DNAis reacted with random primers and a DNA polymerase. The polymerase iscapable of displacing the non-template strand of the DNA double strandduring the amplification (e.g., a φ29 polymerase). The displaced strandsserve as a matrix for the extension of further primers. The treatmentcan take place before or in between the enrichment, in particular beforeany digestion of genomic DNA with non-methylation-specific restrictionenzymes, or before linkers are ligated to the resulting fragments, orbefore all fragments are subjected to a methylation-specific restrictionenzyme digestion.

According to each embodiment of the generation of type-A fragments, thesecond type of sample (herein referred as type B) is generated bycomplexity reduction of genomic DNA by a first methylation non-specificrestriction enzyme digestion and a second methylation specificrestriction enzyme digestion. In particular, the enrichment of DNAfragments is carried out according to the above described method II,wherein genomic DNA is digested with non-methylation-specificrestriction enzymes, linkers are ligated to the resulting fragments, allfragments are subjected to a methylation-specific restriction enzymedigestion, and undigested fragments with linkers on both ends areamplified by linker mediated PCR.

Such generated DNA fragments derived from test and reference sample arethen subjected to one or more CpG-island-arrays and/or to one or moreoligonucleotide arrays according to the invention as described above. Ifsamples of DNA fragments are subject to the same array, the DNAfragments have to be labeled differentially. If the samples of DNAfragments are subject to different identical arrays, the DNA fragmentsmay have the same label or different ones.

After hybridization of the respective DNA fragments on the complementaryoligonucleotides or nucleic acids of the corresponding arrays, one ormore hybridization signals are detectable, and conclusions are drawnfrom the intensity of these signals with respect to variations in DNAcopy-number, and to changes in the methylation pattern, or both.

Copy-number variations are deduced from signals derived from type A DNAfragment samples of test samples in comparison to those signals derivedfrom type A DNA fragment samples of reference samples.

I) If the signal for a test sample has the same intensity as the signalof the corresponding reference sample, then the genomic DNA region inthe genomic DNA of the test sample is present in the same copy-number asin the genome of the reference sample.

II) If the signal for a test sample has an increased intensity incomparison to the signal of the corresponding reference sample, then thegenomic DNA region in the genomic DNA of the test sample is present athigher copy-numbers then in the genomic DNA of the reference sample. Theamplification of the copy-number is thereby proportional to the increaseof the hybridization signal of the test sample relative to that of thereference sample.

III) If the signal for a test sample has a decreased intensity incomparison to the signal of the corresponding reference sample, then thegenomic DNA region in the genomic DNA of the test sample is absent orpresent at lower copy-numbers then in the genomic DNA of the referencesample. The reduction of copy-number is thereby proportional to thedecrease of the hybridization signal of the test sample relative to thatof the reference sample.

Thereby, in each of the above three cases I-III, the genomic DNA regionis characterized in that it comprises at least parts of thecomplementary sequence of the oligonucleotide or nucleic acid to whichthe respective DNA fragment is hybridized.

Changes in the methylation pattern are deduced from signals derived fromtype B DNA fragment samples of the test sample in comparison to thosesignals derived from type B DNA fragment samples of the correspondingreference sample.

I) If the signal for a test sample has the same intensity as the signalof the corresponding reference sample, then the region of the genomicDNA of the test sample corresponding to the hybridized DNA fragmentcomprises the same ratio of methylated to unmethylated cytosines in aCpG context as the corresponding region of the genomic DNA of thereference sample.

II) If the signal for a test sample has an increased intensity incomparison to the signal of the corresponding reference sample, then theregion of the genomic DNA of the test sample corresponding to thehybridized DNA fragment comprises an increased ratio of methylated tounmethylated cytosines in a CpG context compared to the correspondingregion of the genomic DNA of the reference sample. The increase in theratio is thereby proportional to the increase of the hybridizationsignal of the test sample relative to that of the reference sample.

III) If the signal for a test sample has an decreased intensity incomparison to the signal of the corresponding reference sample, then theregion of the genomic DNA of the test sample corresponding to thehybridized DNA fragment comprises a decreased ratio of methylated tounmethylated cytosines in a CpG context compared to the correspondingregion of the genomic DNA of the reference sample. The decrease in theratio is thereby proportional to the decrease of the hybridizationsignal of the test sample relative to that of the reference sample.

Thereby, in each of the above three cases I-III, the genomic DNA regionis characterized in that it comprises at least parts of thecomplementary sequence of the oligonucleotide or nucleic acid to whichthe respective DNA fragment is hybridized.

Embodiment II

A preferred embodiment comprises:

-   -   generating two types of samples of DNA fragments each derived        from a test sample and/or a reference sample;    -   generating the first type of sample comprising a first        methylation-non-specific restriction enzyme digestion and a        second methylation-specific restriction enzyme digestion;    -   generating the second type of sample comprising a        methylation-specific restriction enzyme digestion;    -   deducing an alteration in DNA methylation by comparison of        signal intensities of hybridizations or non-hybridizations        derived form the first type of DNA fragments of the test sample        with those derived from the reference sample, or by comparison        of signal intensities of hybridizations or non-hybridizations        derived from the second type of DNA fragments of a test sample        with those derived from a reference sample, or both; and    -   deducing a copy-number variation by considering a comparison of        signal intensity of hybridizations or non-hybridizations derived        from the first type DNA fragments of a test sample with those        derived from a reference sample and a comparison of signal        intensity of hybridizations or non-hybridizations derived from        the second type DNA fragments of a test samples with those        derived from a reference sample.

Another preferred embodiment comprises a comparison of signalintensities derived from the first type of DNA fragments of a testsample with those derived from a reference sample, wherein:

-   -   the same signal intensity for a test sample and for a reference        sample indicates that the genomic region of the test sample        corresponding to the hybridized DNA fragments comprises the same        ratio of methylated to unmethylated cytosines as the        corresponding genomic region of the reference sample;    -   an increased signal intensity for a test sample in comparison to        a reference sample indicates that the genomic region of the test        sample corresponding to the hybridized DNA fragments comprises        an increased ratio of methylated to unmethylated cytosines        compared to the corresponding genomic region of the reference        sample, wherein the ratio increase is thereby proportional to        the signal increase; and    -   a decreased signal intensity for a test sample in comparison to        a reference sample indicates that the genomic region of the test        sample corresponding to the hybridized DNA fragments comprises a        decrease ratio of methylated to unmethylated cytosines compared        to the corresponding genomic region of the reference sample,        wherein the ratio decrease is thereby proportional to the signal        decrease.

A further preferred embodiment comprises a comparison of signalintensities derived from the second type of DNA fragments of a testsample with those derived from a reference sample, wherein:

-   -   the same signal intensity for a test sample and for a reference        sample indicates that the genomic region of the test sample        corresponding to the hybridized DNA fragments comprises the same        ratio of unmethylated to methylated cytosines as the        corresponding genomic region of the reference sample;    -   an increased signal intensity for a test sample in comparison to        a reference sample indicates that the genomic region of the test        sample corresponding to the hybridized DNA fragments comprises        an increased ratio of unmethylated to methylated cytosines        compared to the corresponding genomic region of the reference        sample, wherein the ratio increase is thereby proportional to        the signal increase; and    -   a decreased signal intensity for a test sample in comparison to        a reference sample indicates that the genomic region of the test        sample corresponding to the hybridized DNA fragments comprises a        decrease ratio of unmethylated to methylated cytosines compared        to the corresponding genomic region of the reference sample,        wherein the ratio decrease is thereby proportional to the signal        decrease.

Another preferred embodiment comprises a comparison of signalintensities derived from the first type DNA fragments of a test samplewith those derived from a reference sample and a comparison of signalintensities derived from second type DNA fragments of a test sample withthose derived from a reference sample, wherein:

-   -   a deletion of a genomic DNA region is indicated by a decreased        signal intensity of first type DNA fragments of a test sample in        comparison to that of a reference sample and a decreased signal        intensity of second type DNA fragments of a test sample in        comparison to that of a reference sample;    -   an amplification of a genomic DNA region is indicated by:

i) an increased signal intensity of DNA fragments of a test sample incomparison to those of a completely methylated reference sample in caseof enrichment of methylated DNA;

ii) an increased signal intensity of DNA fragments of a test sample incomparison to those of a completely unmethylated reference sample incase of enrichment of unmethylated DNA;

iii) an increased signal intensity of DNA fragments of a completelymethylated test sample in comparison to those of a completely methylatedreference sample in case of enrichment of methylated DNA, or

iv) an increased signal intensity of DNA fragments of a completelyunmethylated test sample in comparison to those of a completelyunmethylated reference sample in case of enrichment of unmethylated DNA.

According to embodiment II, two types of samples of DNA fragments aregenerated each from genomic DNA of test and of reference samples. Thefirst type of sample (herein referred as type-B) is generated by a firstmethylation-non-specific restriction enzyme digestion and a secondmethylation-specific restriction enzmye digestion. In particular, type-BDNA fragments are generated according to the above described method II,wherein genomic DNA is digested with non-methylation-specificrestriction enzymes, linkers are ligated to the resulting fragments, allfragments are subjected to a methylation-specific restriction enzymedigestion, and undigested fragments with linkers on both ends areamplified by linker mediated PCR. The second type of sample (hereinreferred to as type C) is generated by a methylation-specificrestriction enzyme digestion. In particular, type C DNA fragments aregenerated according to the above described method I, wherein genomic DNAis digested with methylation-specific restriction enzymes, linkers areligated to the resulting fragments, and fragments with linkers areamplified by linker-mediated PCR.

Such generated DNA fragments derived from test and reference samples arethen subjected to one or more CpG-island-arrays and/or to one or moreoligonucleotide arrays according to the invention as described above. Ifsamples of DNA fragments are subject to the same array, the DNAfragments have to be labeled differentially. If the samples of DNAfragments are subject to different identical arrays, the DNA fragmentsmay have the same label or different ones.

After hybridization of the respective DNA fragments on the complementaryoligonucleotides or nucleic acids of the corresponding arrays, one ormore hybridization signals are detectable. According to the invention,conclusions are drawn from the intensity of these signals with respectto the variations in the DNA copy-number, and to changes in themethylation pattern, or both.

Changes in the methylation pattern are deduced from signals derived fromtype-B DNA fragment samples of the test sample in comparison to thosesignals derived from type-B DNA fragment samples of the correspondingreference sample.

I) If the signal for a test sample has the same intensity as the signalof the corresponding reference sample, then the region of the genomicDNA of the test sample corresponding to the hybridized DNA fragmentcomprises the same ratio of methylated to unmethylated cytosines in aCpG context as the corresponding region of the genomic DNA of thereference sample.

II) If the signal for a test sample has an increased intensity incomparison to the signal of the corresponding reference sample, then theregion of the genomic DNA of the test sample corresponding to thehybridized DNA fragment comprises an increased ratio of methylated tounmethylated cytosines in a CpG context compared to the correspondingregion of the genomic DNA of the reference sample. The increase in theamount of methylated cytosines is thereby proportional to the increaseof the hybridization signal of the test sample relative to that of thereference sample.

III) If the signal for a test sample has a decreased intensity incomparison to the signal of the corresponding reference sample, then theregion of the genomic DNA of the test sample corresponding to thehybridized DNA fragment comprises a decreased ratio of methylated tounmethylated cytosines in a CpG context compared to the correspondingregion of the genomic DNA of the reference sample. The decrease in theamount of methylated cytosines is thereby proportional to the decreaseof the hybridization signal of the test sample relative to that of thereference sample. Thereby, in each of the three cases I-III, the genomicDNA region is characterized in that it comprises at least parts of thecomplementary sequence of the oligonucleotide or nucleic acid to whichthe respective DNA fragment is hybridized.

Alternatively, changes in the methylation pattern can also be deducedfrom signals derived from type-C DNA fragment samples of the test samplein comparison to those signals derived from type-C DNA fragment samplesof the corresponding reference sample:

I) If the signal for a test sample has the same intensity as the signalof the corresponding reference sample, then the region of the genomicDNA of the test sample corresponding to the hybridized DNA fragmentcomprises the same ratio of unmethylated to methylated cytosines in aCpG context as the corresponding region of the genomic DNA of thereference sample.

II) If the signal for a test sample has an increased intensity incomparison to the signal of the corresponding reference sample, then theregion of the genomic DNA of the test sample corresponding to thehybridized DNA fragment comprises a increased ratio of unmethylated tomethylated cytosines in a CpG context compared to the correspondingregion of the genomic DNA of the reference sample. The increase in theratio is thereby proportional to the increase of the hybridizationsignal of the test sample relative to that of the reference sample.

III) If the signal for a test sample has a decreased intensity incomparison to the signal of the corresponding reference sample, then theregion of the genomic DNA of the test sample corresponding to thehybridized DNA fragment comprises a decreased ratio of unmethylated tomethylated cytosines in a CpG context compared to the correspondingregion of the genomic DNA of the reference sample. The decrease in theratio is thereby proportional to the decrease of the hybridizationsignal of the test sample relative to that of the reference sample.

Thereby, in each of the three cases I-III, the genomic DNA region ischaracterized in that it comprises at least parts of the complementarysequence of the oligonucleotide or nucleic acid to which the respectiveDNA fragment is hybridized.

Of course, the methylation pattern can also be deduced by taking intoaccount the above said for both type-B and type-C DNA fragments.

Copy-number variations are deduced by taking into account signalsderived from type-B DNA fragment samples of the test sample incomparison to those signals derived from type-B DNA fragment samples ofthe corresponding reference sample and signals derived from type C-DNAfragment samples of the test samples in comparison to those signalsderived from type-C DNA fragment samples of the corresponding referencesample.

A deletion of a genomic DNA region is present in the genome of a testsample, if the following two cases apply simultaneously to the same orto different identical oligonucleotides or nucleic acids on arrays: 1)The signal of type-B DNA fragments for a test sample has a decreasedintensity in comparison to the signal of the corresponding referencesample, and 2) the signal of type-C DNA fragments for a test sample hasa decreased intensity in comparison to the signal of the correspondingreference sample.

The amplification of a genomic region can be determined according to thefollowing: First, only the reference sample is ‘treated’ for thedetermination of an amplification, depending on the type of enrichment.In case of the enrichment of methylated DNA, an aliquot of a referencesample is treated appropriately so that all cytosines of CpGdinucleotides are methylated (100% methylated reference sample). Such atreatment can be any kind of treatment, preferably a treatment with amethyltransferase, in particular a treatment with the methyltransferaseSssI. The treatment can take place before or in between the enrichmentaccording to method II, in particular before any digestion of genomicDNA with non-methylation-specific restriction enzymes, or before linkersare ligated to the resulting fragments, or before all fragments aresubjected to a methylation-specific restriction enzyme digestion. Incase of the enrichment of unmethylated DNA, an aliquot of referencesample is treated appropriately so that all cytosines of CpGdinucleotides are unmethylated (0% methylated reference sample). Such atreatment can be any kind of chemical or enzymatic treatment, inparticular the genomic DNA is subject to amplification as well known tothose skilled in the art. Such an amplification is preferably carriedout according to whole genome amplification methods as described above(see section IV “Labeling”). Most preferably the isothermal “MultipleDisplacement Amplification” (MDA) is used. The DNA is reacted withrandom primers and a DNA polymerase. The polymerase is capable ofdisplacing the non-template strand of the DNA double strand during theamplification (e.g., a φ29 polymerase). The displaced strands serve as amatrix for the extension of further primers.

An amplification of a genomic DNA region is present in the genome of atest sample, i) if the signal for a test sample has an increasedintensity in comparison to the signal of the corresponding 100%methylated reference sample (in case of enrichment of methylated DNA) orii) if the signal for a test sample has an increased intensity incomparison to the signal of the corresponding 0% methylated referencesample (in case of the enrichment of unmethylated DNA). In either case,the genomic DNA region is present at higher copy-numbers in the genomicDNA of the test sample compared to the genomic DNA of the referencesample. The amplification of the copy-number is thereby proportional tothe increase of the hybridization signal relative to the 100% methylatedreference sample or the 0% methylated reference sample, as applicable.

Second, it is also possible to treat an aliquot of the test sample inaddition to an aliquot of the reference sample according to theenrichment of choice. For enrichment of methylated DNA, an aliquot of atest sample and an aliquot of a reference sample are treatedappropriately so that all cytosines of CpG dinucleotides are methylated(100% methylated test sample and 100% methylated reference sample). Asexplained, such a treatment can be any kind of treatment, preferably atreatment with a methyltransferase, in particular a treatment with themethyltransferase SssI. Again, the treatment can take place before or inbetween the enrichment according to method II, in particular before anydigestion of genomic DNA with non-methylation-specific restrictionenzymes, or before linkers are ligated to the resulting fragments, orbefore all fragments are subjected to a methylation-specific restrictionenzyme digestion. For enrichment of unmethylated DNA, an aliquot of testsample and an aliquot of a reference sample are treated appropriately sothat all cytosines of CpG dinucleotides are unmethylated (0% methylatedtest sample and 0% methylated reference sample). As already explained,such a treatment can be any kind of chemical or enzymatic treatment, inparticular the genomic DNA is subject to amplification according toart-recognized methods. Such an amplification is preferably carried outaccording to whole genome amplification methods as described above (seesection IV “Labeling”). Most preferably the isothermal “MultipleDisplacement Amplification” (MDA) is used. The DNA is reacted withrandom primers and a DNA polymerase. The polymerase is capable ofdisplacing the non-template strand of the DNA double strand during theamplification (e.g. a φ29 polymerase). The displaced strands serve as amatrix for the extension of further primers.

In this case, an amplification of a genomic DNA region is present in thegenome of a test sample, i) if the signal for the 100% methylated testsample has an increased intensity in comparison to the signal of thecorresponding 100% methylated reference sample (in case of enrichment ofmethylated DNA) or ii) if the signal for the 0% methylated test samplehas an increased intensity in comparison to the signal of thecorresponding 0% methylated reference sample (in case of enrichment ofunmethylated DNA). In either case, the genomic DNA region is present athigher copy-numbers in the genomic DNA of the test sample compared tothe genomic DNA of the reference sample. The amplification of thecopy-number is thereby proportional to the increase of the hybridizationsignal of the test sample relative to that of the reference sample.

Embodiment III

A preferred embodiment comprises:

-   -   generating DNA fragments derived from a test sample, a        completely methylated aliquot of a reference sample and of a        completely unmethylated aliquot of said reference sample by        enrichment of methylated DNA;    -   obtaining a value represented by the quotient of the difference        of the signal intensity of the test sample and the signal        intensity of the completely unmethylated reference sample to the        difference of the completely methylated reference sample and the        completely unmethylated reference sample;    -   deducing that values larger than 1 represent an increase of the        copy-number of the analyzed genomic region in the test sample.        A further preferred embodiment comprises:    -   generating DNA fragments derived from a test sample, a        completely methylated aliquot of a reference sample and of a        completely unmethylated aliquot of said reference sample by        enrichment of unmethylated DNA;    -   obtaining a value represented by the quotient of the difference        of the signal intensity of the test sample and the signal        intensity of the completely methylated reference sample to the        difference of the completely unmethylated reference sample and        the completely methylated reference sample; and    -   deducing that values larger than 1 represent an increase of the        copy-number of the analyzed genomic region in the test sample.

According to this embodiment, genomic DNA derived from a referencesample is subject to two different treatments. On the one hand analiquot of the genomic DNA of a reference sample is treated so thatcompletely unmethylated DNA is generated (0% methylated referencesample). Such a treatment can be of any suitable chemical or enzymatictreatment, in particular the genomic DNA is subject to amplification asis well known in the art. Such amplification is preferably carried outaccording to whole genome amplification methods as described above (seesection IV “Labeling”). Most preferably the isothermal “MultipleDisplacement Amplification” (MDA) is used. The DNA is reacted withrandom primers and a DNA polymerase. The polymerase is capable ofdisplacing the non-template strand of the DNA double strand during theamplification (e.g. a φ29 polymerase). The displaced strands serve as amatrix for the extension of further primers.

On the other hand, an aliquot of the genomic DNA of a reference sampleis treated so that all cytosines of CpG dinucleotides are methylated(100% methylated reference sample). Such a treatment can be of anysuitable treatment, for example a treatment with a methyltransferase, inparticular a treatment with the methyltransferase SssI.

According to this embodiment, methylated or unmethylated DNA is enrichedas described above from the test sample as well as from the 0%methylated and the 100% methylated reference sample. In the following,enriched methylated DNA from the test sample, the 0% methylated and the100% methylated reference sample are compared with each other orenriched unmethylated DNA from the test sample, the 0% methylated andthe 100% methylated reference sample are compared with each other.

Where unmethylated DNA is enriched, the maximal signal will result fromthe 0% methylated reference sample while the minimal signal will resultfrom the 100% methylated sample. On the other hand, where methylated DNAis enriched, the maximal signal will result from the 100% methylatedreference sample while the minimal signal will result from the 0%methylated sample.

Equally, if methylated or unmethylated DNA is enriched, an “X-value,” asreferred to herein, is calculated for each oligonucleotide or nucleicacid of the array or corresponding arrays. Accordingly, this X-value isdefined by the following formula:

$X = \frac{I_{test} - I_{\min}}{I_{\max} - I_{\min}}$

Wherein I_(test) represents the signal intensity obtained for the testsample, I_(min) represents the signal intensity obtained for the 100%methylated reference sample if unmethylated DNA is enriched or for the0% methylated reference sample if methylated DNA is enriched, andI_(max) represents the signal intensity obtained for the 0% methylatedreference sample if unmethylated DNA is enriched or for the 100%methylated reference sample if methylated DNA is enriched.

In case, X-values larger than “1” are obtained, an amplification of thecorresponding region in the genome of the test sample has occurred. Todetermine the actual copy number, the above-described embodiments I orII has to be performed. With knowledge of the copy number, a person ofordinary skill in the art will know how to interpret the signalintensity for the corresponding fragments of said genomic region and tocalculate the percentage of methylation (see section V) from the signalintensity.

VII. Quantification of Signal Intensities

In particular embodiments, the signal intensities are not quantified.However, it may be favourable in certain cases to quantify the signalintensities. Therefore, in a preferred embodiment, signal intensitiesderived by the inventive means are quantified. Such a quantification canbe performed by suitable methods familiar to those of ordinary skill inthe art.

In a particular preferred embodiment, quantification is performed asfollows: Genomic DNA derived from a reference sample is subject to twodifferent treatments. On the one hand, an aliquot of the genomic DNA ofa reference sample is treated so that completely unmethylated DNA isgenerated (0% methylated reference sample). Such a treatment can be ofany suitable chemical or enzymatic treatment, in particular the genomicDNA is subject to amplification as familiar in the art. Such anamplification is preferably carried out according to whole genomeamplification methods as described above (see section IV, “Labeling”).Most preferably the isothermal “Multiple Displacement Amplification”(MDA) is used. The DNA is reacted with random primers and a DNApolymerase. The polymerase is capable of displacing the non-templatestrand of the DNA double strand during the amplification (e.g. a φ29polymerase). The displaced strands serve as a matrix for the extensionof further primers.

On the other hand, an aliquot of the genomic DNA of a reference sampleis treated so that all cytosines of CpG dinucleotides are methylated(100% methylated reference sample). Such a treatment can be of anysuitable treatment, for example a treatment with a methyltransferase, inparticular a treatment with the methyltransferase SssI.

According to this embodiment, methylated or unmethylated DNA is enrichedas described above from genomic DNA of the test sample as well as fromthe said 0% methylated and the said 100% methylated reference samples.In the following, enriched methylated DNA from the test sample, from the0% methylated and from the 100% methylated reference sample are takeninto account, or enriched unmethylated DNA from the test sample, fromthe 0% methylated and from the 100% methylated reference sample aretaken into account. Of course it is possible, but not necessary, tocalculate the mean value and other statistically relevant values fromthe results obtained for methylated and unmethylated DNA.

DNA fragments derived from genomic DNA isolated from test and referencesamples are labeled and hybridized to a DNA-microarray, in particular anoligonucleotide microarray according to the invention as described above(see section I, II, lIl and IV). Of course more than two samples can becompared simultaneously if distinguishable labels are used.

According to the invention, hybridization can be performed in differentways. In a particularly preferred embodiment, different identical arraysare used, on each array only DNA fragments derived from genomic DNA of asingle sample are hybridized.

In another particularly preferred embodiment, each sample of DNAfragments obtained from genomic DNA of different samples is labeleddifferentially. Thus, the differentially labeled DNA can be applied tothe same array. Moreover, it is preferred that only some of DNA fragmentsamples are labeled differentially and hybridized to the same array. DNAfragments derived from other samples and labeled with the same labelsare hybridized on a different array.

In a further particularly preferred embodiment, DNA fragments derivedfrom one or more test samples are hybridized each to a differentidentical or all to the same array. The data resulting from thesehybridizations is then compared with data obtained previously for DNAfragments of a reference sample.

In case methylated DNA is enriched, the maximal signal will result fromthe 100% methylated reference sample while the minimal signal willresult from the 0% methylated sample.

For determination of the percentage of methylation, an “X-value,” asreferred to herein, is calculated for each oligonucleotide or nucleicacid of the array or corresponding arrays. According to the invention,this X-value is defined by the following formula:

$X = \frac{I_{test}^{M} - I_{0\%}^{M}}{I_{100\%}^{M} - I_{0\%}^{M}}$

Wherein I^(M) _(test) represents the signal intensity obtained for thetest sample by means of methylated DNA enrichment, I^(M) _(0%)represents the signal intensity obtained for the 0% methylated referencesample by means of methylated DNA enrichment, and I^(M) _(100%)represents the signal intensity obtained for the 100% methylatedreference sample by means of methylated DNA enrichment.

Where unmethylated DNA is enriched, the maximal signal will result fromthe 0% methylated reference sample while the minimal signal will resultfrom the 100% methylated sample. For determination of the percentage ofmethylation, an “X-value,” as referred to herein, is calculated for eacholigonucleotide or nucleic acid of the array or corresponding arrays.According to the invention, this X-value is defined by the followingformula:

$X = {1 - \frac{I_{test}^{UM} - I_{100\%}^{UM}}{I_{0\%}^{UM} - I_{100\%}^{UM}}}$

Wherein I^(UM) _(test) represents the signal intensity obtained for thetest sample by means of unmethylated DNA enrichment, I^(UM) _(100%)represents the signal intensity obtained for the 100% methylatedreference sample by means of unmethylated DNA enrichment, and I^(UM)_(0%) represents the signal intensity obtained for the 0% methylatedreference sample by means of unmethylated DNA enrichment.

In either case, the X-value is a number from which the degree ofmethylation can be deduced. In case the X-value is “0”, all cytosines ofthe analysed CpG dinucleotides in the corresponding genomic DNA regionare unmethylated. X-values in the range between “0” and “1” multipliedby 100% result in the percentage of methylated cytosines of the analysedCpG dinucleotides. An X-value of “1” represents a 100% methylation ofall cytosines of the analysed CpG dinucleotides in the correspondinggenomic DNA region.

A preferred embodiment comprises:

-   -   generating DNA fragments derived from a test sample, a        completely methylated aliquot of a reference sample and of a        completely unmethylated aliquot of said reference sample by        enrichment of methylated DNA;    -   obtaining a value represented by

$\frac{I_{test}^{M} - I_{0\%}^{M}}{I_{100\%}^{M} - I_{0\%}^{M}}$

wherein I^(M) _(test) represents the signal intensity obtained for thetest sample by means of methylated DNA enrichment, I^(M) _(0%)represents the signal intensity obtained for the 0% methylated referencesample by means of methylated DNA enrichment, and I^(M) _(100%)represents the signal intensity obtained for the 100% methylatedreference sample by means of methylated DNA enrichment; and

-   -   deducing, if said value is i) “0” that all analyzed cytosines in        the corresponding genomic DNA region of the test sample are        unmethylated, ii) in the range between “0” and “1” that the        value multiplied by 100 represents the percentage of methylated        cytosines in the corresponding genomic DNA region of the test        sample, or iii) “1” that all analyzed cytosines in the        corresponding genomic DNA region of the test sample are        methylated.        Another preferred embodiment comprises:    -   generating DNA fragments derived from a test sample, a        completely methylated aliquot of a reference sample and of a        completely unmethylated aliquot of said reference sample by        enrichment of unmethylated DNA;

obtaining a value represented by

$1 - \frac{I_{test}^{UM} - I_{100\%}^{UM}}{I_{0\%}^{UM} - I_{100\%}^{UM}}$

wherein I^(UM) _(test) represents the signal intensity obtained for thetest sample by means of unmethylated DNA enrichment, I^(UM) _(100%)represents the signal intensity obtained for the 100% methylatedreference sample by means of unmethylated DNA enrichment, and I^(UM)_(0%) represents the signal intensity obtained for the 0% methylatedreference sample by means of unmethylated DNA enrichment; and

-   -   deducing, if said value is i) “0” that all analyzed cytosines in        the corresponding genomic DNA region of the test sample are        unmethylated, ii) in the range between “0” and “1” that the        value multiplied by 100 represents the percentage of methylated        cytosines in the corresponding genomic DNA region of the test        sample, or iii) “1” that all analyzed cytosines in the        corresponding genomic DNA region of the test sample are        methylated.

VIII. Further Use of all Embodiments According to the Invention

The embodiments according to the invention are also suitable for thediscovery of targets. Targets are proteins or enzymes, the modulation ofwhich is correlated with defined diseases. By determining such acorrelation, a substance can be selected or generated, which modulatesthe target or target forerunners or target successors (up-stream ordown-stream of the determined target in a biological pathway) such thatthe disease-correlated modulation of the target is terminated. Suchsubstances are then suitable for making pharmaceutical compositions forthe prophylaxis or therapy of the disease.

The method according to the invention is also suitable for the discoveryof response markers. Response markers are regulatory regions of thegenome, for instance silencers, enhancers, promoters, etc., or therespective proteins or enzymes, which are correlated with the effect ornon-effect of a specific chemical therapy of a defined disease. Bydetermining such a correlation and the analysis thereof, then thechances of success for a prospective therapy of a patient can bedetermined and/or a patient-specific therapy can be developed byexclusion of therapeutic measures, for which the patient is anon-responder.

Therefore, the invention further relates to the use of a methodaccording to the invention for identifying a response marker, wherein afirst solution with DNA, which originates from a tissue sample withtissue from a non-responder, is analyzed according to the invention,wherein a second solution with DNA, which originates from a tissuesample of the same tissues type, however from a responder, is analyzedaccording to the invention, wherein the first solution and the secondsolution are simultaneously or successively contacted with the DNAmicroarray and hybridized thereon, wherein such immobilized nucleicacids are selected, to which mainly the fragments of the first solutionor of the second solution are hybridized or not hybridized. By such aselected nucleic acid, DNA fragments are identified, which compriseregulatory and/or coding regions of one or several genes. Thus, thecorresponding proteins, peptides or RNAs are derived.

Additionally, for the above method for the discovery of a target, aknown modulator of the coded protein, peptide or RNA determined asmentioned above can be assigned to the specific indication of thediseased tissue. Therefore, the invention further also comprises the useof a modulator assigned by such a method for preparing a pharmaceuticalcomposition with the specific indication, in particular a specificcancer indication.

In further embodiments, the invention provides for the use of a methodaccording to the invention or of a test kit according to the inventionfor the diagnosis of a disease, for example, a cancer disease. A tissuesample is taken from a patient, which is then processed in aconventional way and subjected to the method using the test kit.

All of the cited documents herein are hereby incorporated by referencein their entireties.

DEFINITIONS

The term “treatment” also comprises the prophylaxis and the follow-uptreatment (e.g. of a tumor not detectable anymore or of a stable tumor).The term “prophylaxis” comprises, in conjunction with the detection, themedical check-up as well. The term “detection” or “diagnosis” and/or“treatment” or “therapy” of a cancer disease comprises as an option alsothe detection and/or treatment of metastases of primary tumors in othertissues.

The term “prognosis” as used herein comprises statements about theprobability of a therapy success or treatment success, and/or statementsabout the aggressiveness of a disease, and/or statements about theassumed life time without the occurrence of further disease symptoms ormetastases and/or about the probability of the necessity of anadditional treatment, and/or about the compatibility of undesired sideeffects.

Suitable targets or nucleic acid sequences coding for suitable targetscan be taken from the documents mentioned in the specification.

The amplification of a fragment of a polynucleic acid can for instancebe performed by means of the PCR technology. With regard to theexperimental details, reference is for instance made to the document WO2003/087774.\

With regard to the definition of a “linker” and its structure, againreference is made to the document WO 2003/087774. As a synonym for theterm linker, the term “adapter” is used herein.

“Oligonucleotides” as referred to herein are nucleic acids having alength of 10 to less than 200, in particular of 20 to 100 or 40nucleotides or base pairs. Oligonucleotides may be connected to asubstrate of a microarray by “spacers” and thus be immobilized. Asspacers, nucleic acids having a length of up to 30 nucleotides or basepairs may be used. Alternatively, spacers may be organic compounds,which are chemically connected to one end of an oligonucleotide and arebound with the opposite end to the substrate. Such compounds are knownto those of ordinary skill in the relevant art.

“Methylation-specific restriction enzymes” or “methylation-sensitiverestriction enzymes” are enzymes that: cut a nucleic acid sequence onlyif the recognition site is either not methylated or hemi-methylated; orthat cut only if the latter is methylated. For restriction enzymes,which specifically cut if the recognition site is not methylated orhemimethylated, the cut will not take place, or with a reducedefficiency, if the recognition site is methylated. For restrictionenzymes, which specifically cut if the recognition site is methylated,the cut will not take place, or with a reduced efficiency, if therecognition site is not methylated. Preferred are methylation-specificrestriction enzymes, the recognition sequence of which contains a CGdinucleotide (for instance cgcg or cccggg). Further preferred for someembodiments are restriction enzymes that do not cut if the cytosine inthis dinucleotide is methylated at the carbon atom C5.

“Non-methylation-specific restriction enzymes” or“non-methylation-sensitive restriction enzymes” are restriction enzymesthat cut a nucleic acid sequence irrespective of the methylation statewith nearly identical efficiency. They are also called“methylation-unspecific restriction enzymes.”

A restriction enzyme generates by cutting a “blunt end,” if the doublestrand of the cut nucleic acid is cut at cutting sites being exactlyopposite to each other, with reference to the double strand. Arestriction enzyme generates by cutting a “sticky end,” if the doublestrand of the cut nucleic acid is cut with the cutting sites not beingexactly opposite to each other, with reference to the double strand, butrather forms an overhang at one strand of the double strand.

A “methylation pattern” of a polynucleic acid designates thecharacterization of the nucleic acid sequence as to which nucleotidesthat are capable of being methylated are in fact methylated, and whichnucleotides that are capable of being methylated are not methylated. Amethylation pattern may be given for defined partial regions of thepolynucleic acid or for the whole polynucleic acid.

A “hypomethylation” of a DNA section exists, for example, if a denseseries of CpG dinucleotides has nearly no methylation.

A “hypermethylation” of a DNA section exists, for example, if a denseseries of CpG dinucleotides has nearly a complete methylation.

A “test kit” is an assembly of at least one chemical, biological and/orphysical kit component, together with an instruction or descriptiondescribing the detection of which disease the test kit is intended.Standard reagents and/or standard curves in any form (printed, stored ona data carrier, link to a database) may also be included in a test kit.

A “DNA microarray” is an arbitrary construct with a substrate orcarrier, on which or in which different nucleic acid species, such asgenes, gene fragments or other oligonucleotides or polynucleotides arearranged, respectively at different defined places assigned to therespective nucleic acid species. Typically, at respectively one placeone nucleic acid species is arranged. There may, however, also be adefined mixture of different nucleic acid species arranged atrespectively one place, where, for example, every place carries adifferent mixture. The nucleic acids may be immobilized, this is howevernot necessarily required, depending on the used substrate or carrier.Examples for microarrays include, but are not limited to: nucleic acidmicroarrays, gene microarrays, microtiter plates with nucleic acidsolutions in the wells, the nucleic acids being immobilized or notimmobilized, and membranes with nucleic acids immobilized thereupon.

Of particular importance for the present method variants is the use ofan “oligonucleotide array” microarray or chip, characterized in thatoligonucleotides having a length of up to under 200 bp are immobilizedon a surface.

A “modulator of a target” is a compound or substance, which eitherinhibits or induces the generation of the target, or reduces orincreases the activity of the generated target, referred to the in vitroor in vivo activity in absence of the substance. A modulator may on theone hand be a substance, modulatingly affecting the development cascadeof the target. On the other hand, a modulator may be a substance thatforms a bond with the generated target, such that further physiologicalinteractions with endogenous substances are at least reduced orincreased. Modulators may also be molecules, which affect and inhibit oractivate the transcription of the target gene. Such molecules may forinstance be polyamides or zinc finger proteins, which preventtranscription by binding to DNA regions of the basal transcriptionmachinery. Transcription modulation may also take place indirectly bythe inhibition of transcription factors, which are essential for thetranscription of the target gene. The inhibition of such transcriptionfactors may, for example, be guaranteed by binding to so-called decoyaptamers.

Modulators may be natural or synthetic molecules that specifically bindto a target or target forerunner or target successor. They may also betarget-specific antibodies, for instance human, humanized andnon-humanized polyclonal or monoclonal antibodies. The term antibodiesfurther includes phage display antibodies, ribozyme display antibodies(covalent fusion between RNA and protein) and RNA display antibodies(produced in vitro). The term also includes antibodies that are modifiedby chimerization, humanization or deimmunization, and specific fragmentsof the light and/or heavy chain of the variable region of basicantibodies of the above type. The production or extraction of suchantibodies with given immunogenes is well known in the art. Alsoincluded are bispecific antibodies, which on the one hand bind to atrigger molecule of an immune effector cell (e.g. CD3, CD16, CD64), andon the other hand to an antigen of the tumor target cell. This will, forexample, cause in the case of such binding, killing of a tumor cell.Modulators may for instance also be suitable target-specific anticalinsand affibodies mimicrying an antibody.

A “specific cancer disease” is an organ-specific cancer disease, such aslung cancer, ovary cancer, scrotal cancer, prostate cancer, pancreascancer, breast cancer, cancer of an organ of the digestive tract, etc.Suitable sequences with regard to all aspects of the present inventionare for instance described in the documents DE 20121979 U1, DE 20121978U1, DE 20121977 U1, DE 20121975 U1, DE 20121974 U1, DE 20121973 U1, DE20121972 U1, DE 20121971 U1, DE 20121970 U1, DE 20121969 U1, DE 20121968U1, DE 20121967 U1, DE 20121966 U1, DE 20121965 U1, DE 20121964 U1, DE20121963 U1, DE 20121961 U1, DE 20121960 U1, DE 10019173 A1, DE 10019058A1, DE 10013847 A1, DE 10032529 A1, DE 10054974 A1, DE 10043826 A1, DE10054972 A1, DE 10037769 A1, DE 10061338 A1, DE 10245779 A1, DE 10164501A1, DE 10161625 A1, DE 10230692, DE 10255104, EP 1268855, EP 1283905, EP1268857, EP 1294947, EP 1370685, EP 1395686, EP 1421220, EP 1451354, EP1458893, EP 1340818, EP 1399589, EP 1478784, WO 2004/035803, and WO2005/001141, all of which are incorporated herein by reference in theirentirety.

The “galenic preparation” of a pharmaceutical composition according tothe invention may be performed in a usual way. As counter-ions for ioniccompounds can for instance be used Na⁺, K⁺, Li⁺ or cyclohexyl ammonium.Suitable solid or liquid galenic preparation forms are for instancegranulates, powders, dragees, tablets, (micro) capsules, suppositories,syrups, juices, suspensions, emulsions, drops or injectable solutions(IV, IP, IM, SC) or fine dispersions (aerosols), transdermal systems,and preparations with protracted release of active substance, for theproduction of which usual means are used, such as carrier substances,explosives, binding, coating, swelling, sliding or lubricating agents,tasting agents, sweeteners and solution mediators. As auxiliarysubstances are named here magnesium carbonate, titanium dioxide,lactose, mannite and other sugars, talcum powder, milk protein, gelatin,starch, cellulose and derivatives, animal and vegetable oils such ascod-liver oil, sunflower oil, peanut oil or sesame oil, polyethyleneglycols and solvents, such as sterile water and mono or multi-valentalcohols, for instance glycerin. A pharmaceutical composition accordingto the invention can be produced by that at least one modulator usedaccording to the invention is mixed in a defined dose with apharmaceutically suitable and physiologically well tolerated carrier andpossibly further suitable active, additional or auxiliary substanceswith a defined inhibitor dose, and is prepared in the desired form ofadministration.

“Response markers” are proteins or enzymes or modifications of a nucleicacid (such as SNP or methylation), which are correlated with thecellular response of a cell to an exogenous substance, in particular atherapeutic substance. Different patients react in different ways to aspecific therapy. This is based on the patient-individual cellularresponses to a therapeutic substance. By a differential analysis ofidentical tissues of different persons, the persons suffering from thesame disease and being treated with the same therapy, however reactingin different ways to the therapy (e.g., by healing processes ofdifferent speeds or different disadvantageous effects such as sideeffects), such response markers can be identified, and on the one handthe (differential) existence of a protein or enzyme or a modification ofthe nucleic acid, but also its absence will qualify it as a responsemarker.

“Repeats” also called “repetitive sequences” or “redundant sequences,”are sequences, which are present in many copies in a nucleic acids, forinstance in genomic DNA.

“DMH amplificates” or “amplicons” are DNA fragment mixtures according tothe invention, which were obtained by one or several restrictiondigestions with one or several restriction enzymes according to theinvention, and which were amplified by PCR by means of primers, whereinthe primers hybridize at linkers (adapters), which were ligated after arestriction digestion with one or several restriction enzymes.

EXAMPLES Example 1 Preparation of Two Solutions with One Genomic DNAEach from Two Adjacent Tissue Samples of a Patient

Samples of tumor tissue and adjacent non-neoplastic tissue are obtainedfrom patients that were subjected to a mastectomy. The genomic DNA fromthese tissues is respectively isolated by means of the QIAamp DNA MiniKit (Qiagen, Hilden, Germany) in accordance with the manufacturer'sinstructions. Two preparations are obtained, one with genomic DNA fromdiseased tissue and one with genomic DNA from healthy tissue.

Example 2 Preparation of a DNA Microarray with Oligonucleotides

The definition of the various oligonucleotides for the microarray isperformed as follows: As a database serves the Human Genom EnsemblVersion NCBI 33 database. It is downloaded from the server(www.ensembl.org) in the fasta format. The file contains all availablecontigs of the human genome. By means of software, all fragments arecalculated, which develop (are derivable) by using thenon-methylation-specific restriction enzyme or enzymes that are usedduring the preparation of the solution of fragments. This takes place byrecognition of the respective cutting sites of the restriction enzymesand “in silico” cut. Fragments thus calculated having less than 100 andmore than 1,200 base pairs are (virtually) sorted out. The remainingfragments are tested for cutting sites for the used methylation-specificrestriction enzymes by identifying the corresponding recognitionsequences. Fragments without such recognition sequences are sorted out.For the remaining fragments, further the share of repeats is determined“in silico”. If the share is above 20%, such fragments are sorted out.From the remaining fragments, in an arbitrary manner or according tofurther criteria being not essential for the invention, a number ofpartial sequences are selected as oligonucleotides, which are intendedfor use on the microarray. These oligonucleotide sequences are thensynthesized in a conventional manner on a substrate of a microarray.

Example 3 Preparation of Two Solutions with Fragments of the RespectiveDNA Samples of Example 1 With Methylation-Sensitive Restriction

Section 1: 2 μg each of the genomic DNA of the preparations of Example 1are first fragmented with 5 units each of the non-methylation-specificrestriction enzymes MseI, Bfa1 and Csp6 (available from: New EnglandBiolabs and MBI Fermentas) for 16 hours at 37° C. according tomanufacturer's instructions. Then, these restriction enzymes areinactivated for 20 minutes at 65° C.

Section 2: Thereafter the QiaQuick PCR product purification column kit(Qiagen, Hilden, Germany) is used for purification. According to themanufacturer's information, fragments shorter than 40 bases are veryefficiently removed. It is however not excluded that larger fragments upto a size of approx. 100 bp are also removed hereby. Then, according tothe procedure of Huang et al. (Hum Mol Genet, 8(3):459-470, 1999), aligation of adapters (or linkers) is carried out. For this purpose,different modifications of the original protocol are possible, which aredescribed in the following. For the ligation, the fragmented DNA ismixed with 500 pmol adapter, 400 units T4 DNA Ligase (New EnglandBiolabs), the volume ligase, as recommended by the manufacturer, of 10×buffer and ATP, and incubated for 16 hours at 16° C. The adapters arepreviously prepared by an equimolar mixture of the oligonucleotides H24(5′-AGG CAA CTG TGC TAT CCG AGG GAT-3′) (SEQ ID NO:1) and H12 (5′-TAATCC CTC GGA-3′) (SEQ ID NO:2) is first denaturated for 5 min at 95° C.,and, step by step, cooled down to 25° C. The ligated DNA is finallypurified by means of the QuiaQuick PCR product purification kit (Qiagen,Hilden, Germany).

Thereafter, fragmentation is carried out with 10 units each of themethylation-sensitive (i.e. methylation-specific) restriction enzymesBstU1, HapII, HpyCH4IV and HinP1 (available from: New England Biolabs)for 8 hours at 37° C. and then for 8 hours at 60° C. according tomanufacturer's specification. The fragmented DNA is finally purified bymeans of the QuiaQuick PCR product purification kit (Qiagen, Hilden,Germany).

Approx. 10-100 ng are used in a PCR reaction, which simultaneouslyserves for the amplification of a representation of uncut DNA fragmentsof the order of 50-1,000 bp. The PCR reaction batch contains 350 μMdNTPs, 2.5 μM labelled primer (H24), 5 units DeepVent (exo-) DNApolymerase, 10 μl 10× buffer and 5% DMSO in a volume of 100 μl. Theamplified DNA is finally purified by means of the QuiaQuick PCR productpurification kit (Qiagen, Hilden, Germany).

10-12 PCR reactions are carried out with each sample, in order to obtaina total amount of 20 μg PCR product after the purification. The purifiedPCR products were fragmented and labeled according to the specificationin the “Gene Chip Mapping Assay Manual” of Affymetrix Inc., inparticular chapter 4 (page 38-42).

Thus samples of diseased and of adjacent healthy tissue are obtained,which are suitable for hybridization with the oligonucleotide microarrayof Example 2 or the DNA microarray of Example 8.

Example 4 Hybridization of the Samples on the DNA Microarray

The two solutions obtained in Example 3 (or in Example 9 below) are eachhybridized with a DNA microarray according to Example 2. Hybridizationand detection take place according to the specification in the “GeneChip Mapping Assay Manual” of Affymetrix Inc., in particular chapter 4(page 44-45), and chapter 6 “Washing, Staining & Scanning” (page 75-92).

Two methylation patterns are obtained, and from a comparison of the twomethylation patterns, differences between diseased and healthy tissuecan be recognized. With regard to the differences, the respectiveoligonucleotide set of the DNA microarray is identified and assigned, ifapplicable, to one or several proteins, peptides or RNAs. Normally,these proteins, peptides or RNAs are then differentially expressed inthe respective patient. For the identification of a characteristicmethylation pattern or for the detection of a marker (response ordiagnosis marker), it is however not necessary to build up such acorrelation.

If the differential methylation and the differential expression affectedthereby is confirmed for other patients also having the same disease,and if applicable corresponding cell lines, then the respectiveexpression product is a suitable target for searching substancesinhibiting or inducing the expression product (depending on whether thedifferential expression “diseased”/“healthy” is greater or smaller than1).

Example 5 Comparison of the Fragment Length Distribution of a FragmentMixture Prepared According to the Invention to the Prior Art FragmentLength Distribution

According to the invention, a mixture of fragments of a polynucleic acidaccording to Example 3, Section 1 was prepared. For comparison, themethod according to the document Huang et al., see above, was performed,however with slight modifications. For instance, for a bettercomparison, the same amount of methylation-specific restriction enzymeswas used in both methods. For both fragment solutions, then a fragmentlength histogram was prepared.

The comparative results are shown in FIGS. 1 and 2. In both figures, thefragment length distributions for fragments without methylation-specificrestriction sites are shown by broken lines. The continuous lines showthe fragment length distributions for fragments withmethylation-specific restriction sites. The latter are particularlyinteresting for the further analysis. FIG. 1 shows the results accordingto the prior art. FIG. 2 shows the results of the inventive method, i.e.using several methylation-unspecific restriction enzymes.

From a comparative analysis of FIGS. 1 and 2, it can be seen that withthe inventive method the share of fragments without methylation-specificrestriction sites and having a fragment length below 100 bp iscomparatively high. Consequently, by separation or non-amplification ofsuch short fragments, the non-interesting fragments are selectivelyeliminated. The relative complexity of a fragment solution preparedaccording to the invention is therefore reduced. The number of nucleicacids (in the size window used for the analysis) has decreased, andsimultaneously the percentage (share) of fragments above 100 bpincluding methylation-specific restriction sites has increased.

On the other hand, using the inventive method, the CpG-rich regions thatare interesting for further analysis are cut comparatively shorter, thustheir amplification and detection are substantially facilitated.Furthermore, the probability that all CpG's in the fragments areco-methylated is increased.

Example 6 Hybridization with DMH Amplificates of DNA from PeripheralBlood Lymphocytes (PBL) and from the Breast Cancer Cell Line MDA-Mb-231Demonstrated Inter and Intra Workflow Reproducibility

In order to evaluate the optimized inventive method, DMH amplificates ofDNA from peripheral blood lymphocytes (PBL) and from the breast cancercell line MDA-MB-231 were each prepared twice. These DMH amplificatesolutions were divided up, and hybridization samples were generatedtherefrom, which were hybridized on the specifically prepared Affymetrixmicroarrays. FIGS. 5A and 5B illustrate the inter and intra workflowreproducibility of 0.93-0.95.

Example 7 Potential Marker Candidates that are Differentially MethylatedBetween PBL and Breast Cancer Cell Lines were Reproducibly Identified

247 potential marker candidates being differentially methylated betweenPBL and breast cancer cell lines were reproducibly identified (FIG. 5).Fragments were identified as potential candidates if the log 2difference of the average hybridization signal (log 2 fold change)between two DNA samples was greater than 0.5 (above and below the redline FIG. 5A). For validating these marker candidates, 111 fragments(from the 247) were randomly selected from three groups, which could beseparated according to their log 2 fold change differences (>0.6,0.4−0.6 and <0.4), and were subjected to a direct bisulfite sequencing.

This validation confirmed a high correlation coefficient of 0.71 betweenlog 2 fold change differences and methylation values, which wereobtained by the direct bisulfite sequencing (FIG. 6).

Example 8 Preparation of a DNA Microarray with CpG-Rich DNA Fragments

A DNA microarray is prepared according to the instructions of thedocument Yan et al., Cancer Res., 61:8375-8380, 2001.

Example 9 Preparation of Two Solutions with Fragments of the RespectiveDNA Samples of Example 1 Without Non-Methylation-Sensitive RestrictionEnzymes

2 μg each of the genomic DNA of the preparations of Example 1 arefragmented with 10 units of the methylation-sensitive restrictionendonuclease HapII (New England BioLabs) for 16 hours at 37° C.according to manufacturer's specification, and then the Enzyme isinactivated for 20 minutes at 65° C.

After the restriction, according to the procedure of Huang et al (HumMol Genet, 8(3):459470, 1999), a ligation of adapters, and subsequentPCR amplification of the fragmented DNA by means of theses adapters isperformed. For this purpose, different modifications of the originalprotocol are necessary, which are described in the following.

For the ligation, the fragmented DNA is mixed with 500 pmol adapter, 400units T4 DNA ligase (New England Biolabs), the volume ligase, asrecommended by the manufacturer, of 10× buffer and ATP, and incubatedfor 16 hours at 16° C. The adapters are previously prepared by that anequimolar mixture of the oligonucleotides H24 (5′-AGG CAA CTG TGC TATCCG AGG GAT-3′) (SEQ ID NO:1) and H12-M (5′-CGA TCC CTC GGA-3′) (SEQ IDNO:2) is first denaturated for 5 min at 95° C. and step by step cooleddown to 25° C.

The ligated DNA is purified by means of the QuiaQuick PCR productpurification kit (Qiagen, Hilden, Germany). Approx. 10-100 ng are usedin a PCR reaction, which simultaneously serves for the amplification ofa representation of DNA fragments in the order of 50-1,000 bp, and forlabelling with different fluorescence labels the PCR products of the DNAsamples, which were generated from diseased and from adjacent healthytissue.

The PCR reaction batch for the DNA sample of the diseased tissuecontains 350 μM dNTPs, 0.7 μl Cy5-CTP (Amersham) or in the case of theDNA sample of the healthy tissue Cy3-CTP (Amersham), 2.5 μM Cy5 labelledprimer H24 (or in the case of the DNA sample of the healthy tissue Cy3labelled primer H24), 5 units DeepVent (exo-) DNA polymerase, 10 μl 10×buffer and 5% DMSO in a volume of 100 μl. Of course, the DNA sample ofthe diseased tissue can also be labelled with Cy3, and that of thehealthy tissue with Cy5.

Thus samples of healthy and of adjacent diseased tissue are obtained,which are suitable for hybridization with the oligonucleotide microarrayof Example 2 or the DNA microarray of Example 8.

Example 10 Hybridization of the Samples on the DNA Microarray

The two solutions obtained in Example 3 or in Example 9 are eachhybridized with a DNA microarray according to Example 8. Hybridizationand detection take place according to the Huang et al, Hum Mol Genet,8(3):459-470, 1999. The two differently labelled samples are contactedsimultaneously or subsequently with the DNA microarray and hybridizedthereon. Immobilized nucleic acids, which mainly bind fragments eitherof the sample of the healthy tissue or of the diseased tissue, indicatemethylation differences. In the case of such a different hybridizationbehavior, the respective clone of the DNA microarray is identified andcan be assigned, if applicable, to one or several proteins, peptides orRNAs. Normally, such an expression product is then differentiallyexpressed at the respective patient. If the differential expression isconfirmed for other patients also having the same disease, and ifapplicable corresponding cell lines, then the respective expressionproduct is a suitable target for searching substances inhibiting orinducing the expression product (depending on whether the differentialexpression “diseased”/“healthy” is greater or smaller than 1).

Example 11 DMH Analysis of Neoplastic Breast Tissue

FIG. 3 illustrates the general method of the DMH (differentialmethylation hybridization). FIG. 3 illustrates the differences of theworking procedures (workflow) of the preparation of a mixture ofmethylated fragments of a sample containing DNA. These fragments arereferred to herein as DMH amplificates. The prior art method accordingto Huang et al. is compared here as an example to a method optimizedaccording to the present invention. Both methods for detectingmethylation differ not only in the preparation of the fragments, butalso in the final detection platform. In the method modified accordingto the invention a specifically adapted (customized) Affymetrixoligonucleotide microarray is used as a detection platform. This chipcarries 80,000 oligonucleotides representing approx. 9,000 of the DNAfragments, which were prepared by the DMH method optimized according tothe present invention. The prior art method for preparing the fragments(DMH workflow) generates a solution of great complexity (high number ofgenomic base pairs that are represented by DMH amplificates). Sincemicroarray hybridizations with DMH amplificates of high complexityresult in low signal/noise ratios, the technical object is the provisionof a solution of fragments with reduced complexity, without losinginformative amplificates (e.g. by elimination of repetitive sequences).Further, a high general reproducibility is intended. The solution forthis object is achieved by using the method according to the inventionfor preparing methylated fragments. The complexity of this solution canbe reduced to approx. 5×10⁸ bp, whereas it is 2×10⁹ in the comparableDMH method. This result is illustrated in FIGS. 1 and 2.

DNA samples obtained from “aggressive” and “non-aggressive” tissue werefragmented by a non-methylation-specific restriction hydrolysis (step1). Then adapters were ligated to the fragments, which permitted thesubsequent enzymatic amplification of the fragments (step 2). Theligated fragments were then further digested by a methylation-specificrestriction enzyme hydrolysis (step 3). This restriction was thensubjected to an enzymatic amplification step (step 4) and hybridized onan oligo-DNA chip (step 5). This chip is composed of a bank of detectionoligonucleotides, the design of which is based on an “in silico”digestion of the human genome. Differences in the hybridization patternbetween “aggressive” and “non-aggressive” samples permit theidentification of differentially methylated cutting sites of restrictionenzymes.

1. DNA Isolation.

Samples. Tissue samples were obtained from 17 estrogen receptor-negativefemale patients (Table 1). Breast tumors of 9 patients were rated“aggressive”, since a metastasis occurred in these patients in theobserved period. Tumors of patients, where a metastasis did not occur inthe observed period were rated “non-aggressive”. Three samples ofperipheral blood lymphocytes served as a control.

TABLE 1 Survey of the samples used. Disease- free sur- Total Diseasevival months Age re- Sample type Class (months) survived type currencybreast tumor aggressive 19 39 73 metastasis breast tumor aggressive 1733 78 metastasis breast tumor aggressive 14 41 61 metastasis breasttumor aggressive 16 23 51 metastasis breast tumor aggressive 15 52 46metastasis breast tumor aggressive 9 20 72 metastasis breast tumoraggressive 16 23 52 metastasis breast tumor aggressive 24 25 65metastasis breast tumor aggressive 34 44 69 metastasis breast tumor non-132 132 43 — aggressive breast tumor non- 138 138 73 — aggressive breasttumor non- 128 128 57 — aggressive breast tumor non- 91 91 57 —aggressive breast tumor non- 146 146 68 — aggressive breast tumor non-92 92 64 — aggressive breast tumor non- 129 129 60 — aggressive breasttumor non- 112 112 72 — aggressive peripheral blood n.a. n.a. n.a. n.a.n.a. lymphocytes peripheral blood n.a. n.a. n.a. n.a. n.a. lymphocytesperipheral blood n.a. n.a. n.a. n.a. n.a. lymphocytes * n.a. = notapplicable2. Preparation of the Oligonucleotide Microarray

The sequence of the different oligonucleotides, which were used for theoligonucleotide array, was determined as follows. All sequences, whichwere needed for designing the microarray, originate from the EnsemblHuman Genome Database. The database was downloaded from the server(www.ensembl.org) in the fasta format. The file contains all availablecontigs of the human genome. By means of software, all oligonucleotideswere designed, i.e. “in silico”.

This software simulated the digestion of the human genome first byselected non-methylation-specific restriction enzymes(non-methylation-specific: the restriction is independent from themethylation state of the cutting site) and then by selectedmethylation-specific restriction enzymes. The software then generatesthe sequences of all non-methylation-sensitively digested fragments, apartial amount of these fragments was then selected for the microarray.Fragments of less than 100 and more than 1,200 base pairs were(virtually) rejected. Of the remaining fragments, those were selected,which contained a recognition site of at least one of the previouslyused methylation-specific restriction enzymes. Of these fragments,fragments were further selected, which have up to 20% repeats.

The thus obtained fragments can further be selected either by furthercriteria or randomly. The group of fragments selected at the end werethen synthesized on the surface of the microarray in analogous ways asin the conventional methods.

3. Enzymatic Restriction of the DNA Samples (of Section 1).

The genomic DNA was prepared for the hybridization of the microarray:

Step 1: 2 μg each of an isolated genomic DNA sample was digested with 5units each of the non-methylation-specific restriction enzymes MseI,Bfa1 and Csp6 (available from: New England Biolabs and MBI Fermenters)for 16 hours at 37° C. according to manufacturer's instructions. Then,these restriction enzymes were inactivated by heating for 20 minutes at65° C.

Step 2: Thereafter the QiaQuick PCR product purification column kit(Qiagen, Hilden, Germany) was used for purification. According tomanufacturer's information, fragments shorter than 40 base pairs arethereby removed. It cannot however be excluded that some largerfragments up to a size of 100 base pairs are also removed. Adapters (orlinkers) were then ligated to the fragments. This took place accordingto the procedure described by Huang et al, Hum Mol Genet, 8(3):459-470(1999), and this protocol was adjusted as follows: The fragmented DNAwas mixed with 500 pmol adapter, 400 units T4 DNA Ligase (New EnglandBiolabs), and the volume ligase, as recommended by the manufacturer, of10× ligase buffer and ATP. The incubation was carried out for 16 hoursat 16° C. The adapters were previously prepared by an equimolar mixtureof the oligonucleotides H24 (5′-AGG CAA CTG TGC TAT CCG AGG GAT-3′) (SEQID NO:1) and H12 (5′-TAA TCC CTC GGA-3′) (SEQ ID NO:2) was denaturatedfor 5 min at 95° C. and step by step cooled down to 25° C. Then theligated DNA was purified by means of the QuiaQuick PCR productpurification column kit (Qiagen, Hilden, Germany).

Thereafter, the purified ligated DNA was digested with 10 units each ofthe methylation-sensitive (i.e. methylation-specific) restrictionenzymes BstU1, HapII, HpyCH4IV and HinP1 (available from: New EnglandBiolabs) for 8 hours at 37° C. and then for 8 hours at 60° C. accordingto manufacturer's specification. The fragmented DNA is finally purifiedby means of the QuiaQuick PCR product purification column kit (Qiagen,Hilden, Germany).

Each of the ligated fragments was then amplified, in double reactions.Approx. 10-100 ng were used for a PCR reaction, which amplified onlyuncut DNA fragments in a region of 50-1,000 bp. The PCR reaction batchcontained 350 μM dNTPs, 5 units DeepVent (exo-) DNA polymerase, 10 μl10× buffer and 5% DMSO in a volume of 100 μl. The amplified DNA isfinally purified by means of the QuiaQuick PCR product purificationcolumn kit (Qiagen, Hilden, Germany).

10-12 PCR reactions are carried out with each sample, in order to obtaina total amount of 20 μg PCR product after the purification. The purifiedPCR products are fragmented and labeled according to the specificationin the “Gene Chip Mapping Assay Manual” of Affymetrix Inc., inparticular chapter 4 (page 38-42).

4. Hybridization of the Samples on the Microarray.

As in section 3, labelled amplificates were hybridized on anoligonucleotide microarray according to section 2. Hybridization anddetection took place according to the specification in the “Gene ChipMapping Assay Manual” of Affymetrix Inc., in particular chapter 4 (page44-45), and chapter 6 “Washing, Staining & Scanning” (page 75-92).

Every sample generated an individual hybridization pattern. Thereby,methylation differences between “aggressive” and “non-aggressive” tissueor between peripheral blood lymphocytes and tumor tissue could bederived, by determining DNA fragment sequences, which showed adifferential hybridization signal for the samples of the comparedtissues. Further, it was tried to identify for every identified DNAsequence a corresponding cDNA, which would have as a consequence thatsuch a cDNA would be differentially expressed between the said groups.

The differentially methylated fragments were then subjected to a directbisulfite sequencing, in order to obtain further information with regardto the extent of the methylation.

Example 12 Enrichment of Methylated DNA Fragments by Means of ColumnChromatography

An affinity chromatography column is prepared by immobilizing themethylation-binding domain (MBD) of the protein MeCP2 of the rat by aHis tag on a commercial matrix for the column chromatography. Thepreparation and application of this column were already described inCross et al., Nature Genetics, 1994.

Genomic DNA is first fragmented by ultrasonic treatment and then appliedon the column. Depending on the methylation and the CpG density, the DNAfragments are bound to the column and then collected by elution with aNaCI salt gradient in fractions. Since with increasing saltconcentration, methylated fragments only having a high CpG densityremain bound to the column, they are enriched in the fractions havinghigh salt concentrations.

The enriched methylated DNA fragments are amplified with the BioPrimerLabeling Kit (Invitrogen) and then fragmented and biotin-labeled(GeneChip Mapping 10K Xba Assay Kit, Affymetrix, steps 7 and 8). In thesubsequent hybridization, the methylated DNA fragments are detected.

Example 13 Enrichment of Methylated DNA Fragments by Means of MagneticBeads

For accumulating methylated DNA fragments, magnetic beads are used, onwhich the methylation-binding domain (MBD) of the protein MeCP2 isimmobilized.

Genomic DNA is first fragmented by ultrasonic treatment, then magneticbeads are added. Then the magnetic beads, to which methylated DNAfragments having a high CpG density are bound, are separated, and theselected DNA fragments are separated again from the magnetic beads byincreasing the NaCl concentration.

The enriched methylated DNA fragments are amplified with the BioPrimeLabeling Kit (Invitrogen) and then fragmented and biotin-labeled(GeneChip Mapping 10K Xba Assay Kit, Affymetrix, steps 7 and 8). In thesubsequent hybridization, the methylated DNA fragments are detected.

Example 14 Enrichment of Methylated DNA Fragments by Means ofImmunoprecipitation

Methylated DNA fragments are enriched by immunoprecipitation using amethyl cytosine-binding antibody (Eurogentec). This method has alreadybeen described in Weber et al., Nature Genetics, 2005.

Genomic DNA is first fragmented by ultrasonic treatment. The DNA isdenaturated and then immunoprecipitated. The antibody-bound DNA isseparated by magnetic beads from not-bound DNA and then released.

The enriched methylated DNA fragments are amplified with the BioPrimeLabeling Kit (Invitrogen) and then fragmented and biotin-labeled(GeneChip Mapping 10K Xba Assay Kit, Affymetrix, steps 7 and 8). In thesubsequent hybridization, the methylated DNA fragments are detected.

Example 15 Enrichment of Methylated DNA Fragments by Means of ChromatinImmunoprecipitation

DNA fragments are selected by chromatin immunoprecipitation usingantibodies against transcription factors such as E2F4 (Weinmann et al.,Genes and Development, 2002). By this selection, a series of DNAfragments are obtained, which are bound by a specific transcriptionfactor, and thus fragments, which are located in regulatory regions ofthe genome. In order to investigate the methylation of these fragments,they are provided, as already described (see DMH: linker ligation), withlinkers, methylation-specifically cut and then amplified, fragmented,biotin-labeled (GeneChip Mapping 10K Xba Assay Kit, Affymetrix, steps 7and 8) and hybridized.

Example 16 Enrichment of Non-Methylated DNA Fragments by UsingRestriction Endonucleases Cutting Methylated DNA

Genomic DNA is cut with a restriction endonuclease, which only cuts ifthe DNA is methylated. Such enzymes are McrBC (New England Biolabs), BisI (SibEnzyme) or Gla I (SibEnzyme). These enzymes are used in lieu ofthe methylation-sensitive enzymes in the methylation-specific cutting inthe DMH. In this case, only unmethylated fragments are maintained andare then fragmented and biotin-labeled (GeneChip Mapping 10K Xba AssayKit, Affymetrix, steps 7 and 8) and hybridized.

Example 17 Enrichment of Methylated DNA Fragments by Using RestrictionEndonucleases Cutting Methylated DNA

Further, a use is possible, as already described in Lippman et al.Nature Methods, 2005. Herein, the genomic DNA is first fragmented byultrasonic treatment and then cut with McrBC. DNA fragments, whichexceed a certain size and are thus not cut, are extracted from anagarose gel. The DNA thus obtained is fragmented, biotin-labeled(GeneChip Mapping 10K Xba Assay Kit, Affymetrix, steps 7 and 8) andhybridized.

Example 18 Analysis of the Fragment Length Distribution of a FragmentMixture Prepared According to the Invention

As a database serves the Human Genom Ensembl Version NCBI 33 database.It is downloaded from the server (www.ensembl.org) in the fasta format.The file contains all available contigs of the human genome. By means ofsoftware, all fragments are calculated, which would be derivable byusing the non-methylation-specific restriction enzyme BstU. This takesplace by recognition of the BstU cutting sites and “in silico” cut. Forthe fragments thus obtained the share of CpG islands is determined. Forthis purpose, first CpG islands on the genomic DNA are annotatedaccording to the criterion that in a 200 bp long section, there are atleast 2% CG dinucleotides. In a second step, it is verified whetherfragments, which have been generated by the “in silico” digestion withBstU, are in agreement with the determined CpG islands.

FIGS. 7 and 8 show a fragment/length histogram for fragments with ashare of CpG islands of more than 0.3 (FIG. 7) or of at most 0.3 (FIG.8). The vertical marking in the figures shows respective fragmentshaving a length of 1,000 bp.

As can easily be seen from FIG. 7, the fragments produced by the methodaccording to the invention have for a share of CpG islands of more than0.3 nearly exclusively a length smaller than 1,000 bp. Simultaneously,the fragments produced by the method according to the invention(method 1) with a share of CpG islands of at most 0.3 are mainlyfragments having a length of more than 1,000 bp (FIG. 8).

A database serves the Human Genom Ensembl Version NCBI 33 database. Itis downloaded from the server (www.ensembl.org) in the fasta format. Thefile contains all available contigs of the human genome. By means ofsoftware, all fragments are calculated, which would develop by using thenon-methylation-specific restriction enzyme BstU. This takes place byrecognition of the BstU cutting sites and “in silico” cut. For thefragments thus obtained the share of CpG islands is determined. For thispurpose, first CpG islands on the genomic DNA are annotated according tothe criterion that in a 200 bp long section, there are at least 2% CGdinucleotides. In a second step, it is verified whether fragments, whichhave been generated by the “in silico” digestion with BstU, are inagreement with the determined CpG islands. FIG. 8 shows afragment/length histogram for fragments with a share of CpG islands ofat most 0.3. The vertical marking in the figures shows fragments havinga length of 1,000 bp. As FIG. 8 makes clear, the fragments produced bythe method according to the invention (method I) with a share of CpGislands of at most 0.3 are mainly fragments having a length of more than1,000 bp.

As can be seen from Table 2, the complexity is reduced to 3.8%(1.0×10⁸/2.6×10⁹) of all base pairs of the complete genome. Thefragments obtained thereby can then be used in a PCR amplification inExample 9 and further be analyzed as described there. According thereto,FIG. 7 makes clear that primarily only fragments are amplified, thathave a share of CpG islands of more than 0.3. FIG. 8 shows, however,that the main part of the genome, DNA fragments having a share of atmost 0.3, is separated.

TABLE 2 Fragments Fragments ≦1,000 bps >1,000 bps and and ≧100 bps <100bps Share of CpG islands > 0.3 5.5 × 10⁷ 1.4 × 10⁸ Share of CpG islands≦ 0.3 4.8 × 10⁷ 2.5 × 10⁹ Total DNA 1.0 × 10⁸ 2.6 × 10⁹

Example 19 Preparation of Two Solutions with Fragments of the RespectiveDNA Samples of Example 1 Without Methylation-Sensitive RestrictionEnzymes Suitable to Analyse Copy-Numbers

Section 1: 2 μg each of the genomic DNA of the preparations of Example 1are first fragmented with 5 units each of the non-methylation-specificrestriction enzymes MseI, Bfa1 and Csp6 (available from: New EnglandBiolabs and MBI Fermentas) for 16 hours at 37° C. according tomanufacturer's instructions. Then, these restriction enzymes areinactivated for 20 minutes at 65° C.

Section 2: Thereafter the QiaQuick PCR product purification column kit(Qiagen, Hilden, Germany) is used for purification. According tomanufacturer's information, fragments shorter than 40 bases are veryefficiently removed. It is not excluded, however, that some largerfragments up to a size of approx. 100 bp are also removed thereby. Then,according to the procedure Huang et al (Hum Mol Genet, 8(3):459-470,1999), a ligation of adapters (or linkers) is carried out. For thispurpose, different modifications of the original protocol are possible,which are described in the following. For the ligation, the fragmentedDNA is mixed with 500 pmol adapter, 400 units T4 DNA Ligase (New EnglandBiolabs), the volume ligase, as recommended by the manufacturer, of 10×buffer and ATP, and incubated for 16 hours at 16° C. The adapters arebefore prepared by that an equimolar mixture of the oligonucleotides H24(5′-AGG CAA CTG TGC TAT CCG AGG GAT-3′) and H12 (5′-TAA TCC CTC GGA-3′)is first denaturated for 5 min at 95° C. and step by step cooled down to25° C. The ligated DNA is finally purified by means of the QuiaQuick PCRproduct purification kit (Qiagen, Hilden, Germany).

Approx. 10-100 ng are used in a PCR reaction, which simultaneouslyserves for the amplification of a representation of uncut DNA fragmentsin the order of 50-1,000 bp. The PCR reaction batch contains 350 μMdNTPs, 2.5 μM marked primer (H24), 5 units DeepVent (exo-) DNApolymerase, 10 μl 10× buffer and 5% DMSO in a volume of 100 μl. Theamplified DNA is finally purified by means of the QuiaQuick PCR productpurification kit (Qiagen, Hilden, Germany).

10-12 PCR reactions are carried out with each sample, in order to obtaina total amount of 20 μg PCR product after the purification. The purifiedPCR products were fragmented and labeled according to the specificationin the “Gene Chip Mapping Assay Manual” of Affymetrix Inc., inparticular chapter 4 (page 38-42).

Thus samples of diseased and of adjacent healthy tissue are obtained,which are suitable for hybridization with the oligonucleotide microarrayof Example 2 or the DNA microarray of Example 8.

Example 20 Hybridization of the Samples on the DNA Microarray forAnalysing the Copy Number

The two solutions obtained in example 19 are each hybridized with a DNAmicroarray according to Example 2. Hybridization and detection takeplace according to the specification in the “Gene Chip Mapping AssayManual” of Affymetrix Inc., in particular chapter 4 (page 44-45), andchapter 6 “Washing, Staining & Scanning” (page 75-92).

Two hybridization patterns are obtained, and from a comparison of thetwo patterns, differences of copy numbers between diseased and healthytissue can be recognized. With regard to the differences, the respectiveoligonucleotide set of the DNA microarray is identified and assigned, ifapplicable, to one or several proteins, peptides or enzymes. Normally,these proteins, peptides or enzymes are then differentially expressed atthe respective patient. For the identification of a characteristic copynumber change or for the detection of a marker (response or diagnosismarker), it is however not necessary to build up such a correlation.

If the differential copy numberrs and the differential expressionaffected thereby is confirmed for other patients also having the samedisease, and if applicable corresponding cell lines, then the respectiveexpression product is a suitable target for searching substancesinhibiting or inducing the expression product (depending on whether thedifferential expression “diseased”/“healthy” is greater or smaller than1).

Example 21 Signal Intensity Values for Three Microarray-Chips Each with10 Immobilized Nucleic Acids

After hybridization of DNA on the microarray on respective correlatedimmobilized nucleic acids under defined stringent conditions, thespatially resolved detection of such nucleic acids is performed byscanning for fluorescence radiation emitted by the fluorescence dyewhich is the label of the hybridized DNA. This spatially resolveddetection leads to a signal intensity value for each immobilized nucleicacid. An example for such signal intensity values for threemicroarray-chips each with 10 immobilized nucleic acids is given inTable 3.

TABLE 3 Signal intensity values for three microarray-chips each with 10immobilized nucleic acids. Category of Number of nucleic immobilizedacid nucleic acid chip 1 chip 2 chip 3 Set 1 1 3 4 17 Set 1 2 4 15 18Set 1 3 10 5 16 Set 2 4 14 6 3 Set 2 5 2 9 11 Set 2 6 17 17 19 Set 2 7 93 1 control 8 16 20 12 control 9 11 18 6 control 10 20 7 20

Example 22 Applying the “Log-Transformation” on the Signal IntensityValues of Example 21

The signal intensity values of example 21 were subjected to a“Log-Transformation” according to the following formula:log 2(signal intensity value)

The resulting value is then rounded according to the needed accuracy.For simplicity reasons the resulting value is listed in Table 4 withonly two decimal digits.

TABLE 4 Values resulting after application of the Log-Transformation tothe base 2 for the signal intensity values of Example 21. Category ofNumber of nucleic immobilized acid nucleic acid chip 1 chip 2 chip 3 Set1 1 1.58 2.00 4.09 Set 1 2 2.00 3.91 4.17 Set 1 3 3.32 2.32 4.00 Set 2 43.81 2.58 1.58 Set 2 5 1.00 3.17 3.46 Set 2 6 4.09 4.09 4.25 Set 2 73.17 1.58 0.00 control 8 4.00 4.32 3.58 control 9 3.46 4.17 2.58 control10 4.32 2.81 4.32

Example 23 Applying the “Quantile Normalization” on the“log-transformed” Signal Intensity Values of Example 22

The “log-transformed signal intensity values of Example 22 are subjectedto a “Quantile Normalization”. The result of this operation leads to aequal signal intensity distribution over every consideredmicroarray-chip. The “Quantile Normalization” is done according to thefollowing algorithm:

a) Order the values according to their size for every microarray-chip.Table 4 is thereby transformed into Table 5.

Table 5 shows the result of the first step of the “QuantileNormalization”, the ordering of values according to their size.

chip 1 chip 2 chip 3 1.00 1.58 0.00 1.58 2.00 1.58 2.00 2.32 2.58 3.172.58 3.46 3.32 2.81 3.58 3.46 3.17 4.00 3.81 3.91 4.09 4.00 4.09 4.174.09 4.17 4.25 4.32 4.32 4.32

b). The arithmetic mean value is calculated for each immobilized nucleicacid over the different microarray-chips. The results are illustrated byTable 6.

Table 6 shows the results of the second step of the “QuantileNormalization”, the calculation of the arithmetic mean values.

arithmetic chip 1 chip 2 chip 3 means 1.00 1.58 0.00 0.86 1.58 2.00 1.581.72 2.00 2.32 2.58 2.30 3.17 2.58 3.46 3.07 3.32 2.81 3.58 3.24 3.463.17 4.00 3.54 3.81 3.91 4.09 3.93 4.00 4.09 4.17 4.09 4.09 4.17 4.254.17 4.32 4.32 4.32 0.86

c) Replacing the signal intensity values by their correspondingarithmetic mean value. The results are illustrated by Table 7.

Table 7 shows the results of the third step of the “QuantileNormalization”, the replacing of the signal intensity values by thecorresponding arithmetic mean values.

Chip 1 Chip 2 Chip 3 0.86 0.86 0.86 1.72 1.72 1.72 2.30 2.30 2.30 3.073.07 3.07 3.24 3.24 3.24 3.54 3.54 3.54 3.93 3.93 3.93 4.09 4.09 4.094.17 4.17 4.17 4.32 4.32 4.32

d) Reordering of the values according to their original order on themicroarray-chips. The results are illustrated by Table 8.

Table 8 shows the results of the fourth step of the “QuantileNormalization”, the reordering of the values according to their originalorder on the microarray-chips.

Category of Number of nucleic immobilized acid nucleic acid chip 1 chip2 chip 3 Set 1 1 1.72 1.72 3.93 Set 1 2 2.30 3.93 4.09 Set 1 3 3.24 2.303.54 Set 2 4 3.93 3.07 1.72 Set 2 5 0.86 3.54 3.07 Set 2 6 4.17 4.094.17 Set 2 7 3.07 0.86 0.86 control 8 4.09 4.32 3.24 control 9 3.54 4.172.30 control 10 4.32 3.24 4.32

Example 24 Applying the “Baseline Shift” on the Signal Intensity Valuesof Example 23 after the “Quantile Normalization”

The values after the “Quantile Normalization” are subjected to the“Baseline Shift”. This procedure is carried out by first calculating thearithmetic mean value of the controls for every chip. Subsequently, thismean value is subtracted from each value (see Table 8) of thecorresponding microarray-chip.

The results are illustrated by Table 9.

Category of Number of nucleic immobilized acid nucleic acid chip 1 chip2 chip 3 Set 1 1 −2.26 −2.19 0.65 Set 1 2 1.68 0.02 0.80 Set 1 3 −0.75−1.61 0.26 Set 2 4 −0.05 −0.84 −1.56 Set 2 5 −3.12 −0.37 −0.22 Set 2 60.18 0.18 0.88 Set 2 7 −0.91 −3.05 −2.43 control 8 0.10 0.41 −0.05control 9 −0.44 0.26 −0.99 control 10 0.34 −0.67 1.03

Example 25 Generation of a Representative Value for the Signal IntensityValues of a Set of Immobilized Nucleic Acids

The signal intensity values as they are shown by Table 9 are subjectedto an operation which leads to one representative value for the signalintensity values of a set of immobilized nucleic acids. This is done byselecting the median value from the signal intensity values ofimmobilized nucleic acids of the same set for each microarray-chip. Thisis illustrated by Table 10.

Table 10 shows the result of the generation of a representative valuefor the signal intensity vales of a set of immobilized nucleic acids.

Category of nucleic acid chip 1 chip 2 chip 3 Set 1 −1.68 −1.61 0.65 Set2 −0.48 −0.60 −0.89 control 0.10 0.26 −0.05These preprocessed signal intensity values are then subjected to furtheranalysis which leads to a deduction of the methylation status of thehybridized DNA.

1. A method for determining a DNA methylation pattern, comprising: a)obtaining a solution comprising a sized-biased amplified mixture ofgenomic DNA restriction fragments, wherein the generation of saidmixture comprises an enrichment step using proteins that bindmethylation-specifically to the DNA, and wherein the composition of therestriction fragment mixture depends on the methylation pattern of thegenomic DNA; b) coupling the amplified fragments with a detectable labelto provide a labeled fragment amplificate; c) contacting the labeledfragment amplificate with at least one DNA microarray having a pluralityof different nucleic acids assigned to different respective arraylocations, wherein hybridization of amplificate fragments with thecorresponding assigned nucleic acids takes place under definablestringency, and wherein the assigned nucleic acids are specific forgenomic fragments obtainable by cutting with the restriction enzymesused in a); and d) detecting the label of the hybridized fragmentamplificate using a suitable detection method, wherein determination ofa hybridization pattern of the assigned array locations is afforded tofurther afford determination of the genomic DNA methylation pattern. 2.A method for determining a DNA methylation pattern, comprising: a)obtaining a solution comprising a sized-biased amplified mixture ofgenomic DNA restriction fragments, wherein the generation of saidmixture comprises the use of a triplex-forming molecule, which whenbrought in contact with the DNA, distinguishes between methylated andnon-methylated DNA, and wherein the composition of the restrictionfragment mixture depends on the methylation pattern of the genomic DNA;b) coupling the amplified fragments with a detectable label to provide alabeled fragment amplificate; c) contacting the labeled fragmentamplificate with at least one DNA microarray having a plurality ofdifferent nucleic acids assigned to different respective arraylocations, wherein hybridization of amplificate fragments with thecorresponding assigned nucleic acids takes place under definablestringency, and wherein the assigned nucleic acids are specific forgenomic fragments obtainable by cutting with the restriction enzymesused in a); and d) detecting the label of the hybridized fragmentamplificate using a suitable detection method, wherein determination ofa hybridization pattern of the assigned array locations is afforded tofurther afford determination of the genomic DNA methylation pattern. 3.A method for determining a DNA methylation pattern, comprising: a)obtaining a solution comprising a sized-biased amplified mixture ofgenomic DNA restriction fragments, wherein the generation of saidmixture comprises the use of the MS AP-PCR method, and wherein thecomposition of the restriction fragment mixture depends on themethylation pattern of the genomic DNA; b) coupling the amplifiedfragments with a detectable label to provide a labeled fragmentamplificate; c) contacting the labeled fragment amplificate with atleast one DNA microarray having a plurality of different nucleic acidsassigned to different respective array locations, wherein hybridizationof amplificate fragments with the corresponding assigned nucleic acidstakes place under definable stringency, and wherein the assigned nucleicacids are specific for genomic fragments obtainable by cutting with therestriction enzymes used in a); and d) detecting the label of thehybridized fragment amplificate using a suitable detection method,wherein determination of a hybridization pattern of the assigned arraylocations is afforded to further afford determination of the genomic DNAmethylation pattern.
 4. A method for determining a DNA methylationpattern, comprising: a) generating, by enrichment of methylated DNA, asized-biased amplified mixture of genomic DNA restriction fragmentsobtained from a test sample, wherein the composition of said mixturedepends on the methylation pattern of the genomic DNA of the testsample; b) generating, by enrichment of methylated DNA, a sized-biasedamplified mixture of genomic DNA restriction fragments obtained from acompletely methylated aliquot of a reference sample, wherein thecomposition of said mixture depends on the methylation pattern of thegenomic DNA of the completely methylated aliquot; c) generating, byenrichment of methylated DNA, a sized-biased amplified mixture ofgenomic DNA restriction fragments obtained from a completelyunmethylated aliquot of said reference sample, wherein the compositionof said mixture depends on the methylation pattern of the genomic DNA ofthe completely unmethylated aliquot; d) labeling the fragments of eachof the mixtures of step (a)-(c) to provide labeled fragmentamplificates; e) contacting the labeled fragment amplificates of step(d) with at least one DNA microarray having a plurality of differentnucleic acids assigned to different respective array locations, whereinhybridization of amplificates with the corresponding assigned nucleicacids takes place under definable stringency, and wherein the assignednucleic acids are specific for genomic DNA restriction fragmentsobtainable by step (a), (b) and (c); f) detecting the label of fragmentamplificates hybridized to assigned nucleic acids using a suitabledetection method; g) obtaining at least one value, each value for anassigned nucleic acid, said value being represented by$\frac{I_{test}^{M} - I_{0\%}^{M}}{I_{100\%}^{M} - I_{0\%}^{M}}$ whereinI^(M) _(test) represents the signal intensity obtained for a fragmentamplificate of the test sample, I^(M) _(0%) represents the signalintensity obtained for a respective fragment amplificate of the 0%methylated reference sample, and I^(M) _(100%) represents the signalintensity obtained for a respective fragment amplificate of the 100%methylated reference; and (h) deducing for each of the values obtainedin step (g), if the value for an assigned nucleic acid is i) “0,” thatall analyzed cytosines in the corresponding genomic DNA region of thetest sample are unmethylated, or deducing, if the value for an assignednucleic acid is ii) in the range between “0” and “1,” that the valuemultiplied by 100 represents the percentage of methylated cytosines inthe corresponding genomic DNA region of the test sample, or deducing, ifthe value for an assigned nucleic acid is iii) “1,” that all analyzedcytosines in the corresponding genomic DNA region of the test sample aremethylated, and wherein a DNA methylation pattern is determined.
 5. Amethod for determining a DNA methylation pattern, comprising: a)generating, by enrichment of unmethylated DNA, a sized-biased amplifiedmixture of genomic DNA restriction fragments from a test sample, whereinthe composition of said mixture depends on the methylation pattern ofthe genomic DNA of the test sample; b) generating, by enrichment ofunmethylated DNA, a sized-biased amplified mixture of genomic DNArestriction fragments from a completely methylated aliquot of areference sample, wherein the composition of said mixture depends on themethylation pattern of the genomic DNA of the completely methylatedaliquot; c) generating, by enrichment of unmethylated DNA, asized-biased amplified mixture of genomic DNA restriction fragments froma completely unmethylated aliquot of said reference sample, wherein thecomposition of said mixture depends on the methylation pattern of thegenomic DNA of the completely unmethylated aliquot; d) labeling thefragments of each of the mixtures of step (a)-(c) to provide labeledfragment amplificates; e) contacting the labeled fragment amplificatesof step (d) with at least one DNA microarray having a plurality ofdifferent nucleic acids assigned to different respective arraylocations, wherein hybridization of amplificates with the correspondingassigned nucleic acids takes place under definable stringency, andwherein the assigned nucleic acids are specific for genomic DNArestriction fragments obtainable by step (a), (b) and (c); f) detectingthe label of fragment amplificates hybridized to assigned nucleic acidsusing a suitable detection method; g) obtaining at least one value, eachvalue for an assigned nucleic acid, said value being represented by$1 - \frac{I_{test}^{UM} - I_{100\%}^{UM}}{I_{0\%}^{UM} - I_{100\%}^{UM}}$wherein I^(UM) _(test) represents the signal intensity obtained for thetest sample by means of unmethylated DNA enrichment, I^(UM) _(100%)represents the signal intensity obtained for the 100% methylatedreference sample by means of unmethylated DNA enrichment, and I^(UM)_(0%) represents the signal intensity obtained for the 0% methylatedreference sample by means of unmethylated DNA enrichment; and h)deducing for each of the values obtained in step (g), if the value foran assigned nucleic acid is i) “0,” that all analyzed cytosines in thecorresponding genomic DNA region of the test sample are unmethylated, ordeducing, if the value for an assigned nucleic acid is ii) in the rangebetween “0” and “1,” that the value multiplied by 100 represents thepercentage of methylated cytosines in the corresponding genomic DNAregion of the test sample, or deducing, if the value for an assignednucleic acid is iii) “1,” that all analyzed cytosines in thecorresponding genomic DNA region of the test sample are methylated, andwherein a DNA methylation pattern is determined.
 6. A method fordetermining at least one of the percentage of methylation and therelative copy-number of positions of a test sample DNA, comprising: a)generating, by enrichment of methylated and/or unmethylated DNA, asized-biased amplified mixture of genomic DNA restriction fragments fromDNA derived from a test sample, wherein the composition of said mixturedepends on the methylation pattern of the genomic DNA of the testsample; b) generating, by enrichment of at least one of methylated andunmethylated DNA, a sized-biased amplified mixture of genomic DNArestriction fragments from DNA derived from a reference sample, whereinthe composition of said mixture depends on the methylation pattern ofthe genomic DNA of the reference sample; c) labeling the fragments ofthe mixtures of step (a) and (b) identically or differentially with oneor more detectable labels; d) contacting the labeled fragmentamplificates of step (c) with at least one DNA microarray having aplurality of different oligonucleotides assigned to different respectivearray locations, wherein hybridization of amplificates with thecorresponding assigned oligonucleotides takes place under definablestringency, and wherein the assigned oligonucleotides are specific forgenomic DNA restriction fragments obtainable by step (a) and (b),wherein labeled amplificates are hybridized onto assignedoligonucleotides of the DNA microarray; e) detecting the spatiallyresolved signal intensities of oligonucleotide array locations; and f)comparing the detected signal intensities derived for the amplificatesof the test sample with those derived for the amplificates of thereference sample, wherein the percentage of methylation and/or therelative copy-number of positions of the test sample DNA is deduced. 7.A method for determining a DNA methylation pattern, comprising: a)obtaining a solution comprising a sized-biased amplified mixture ofgenomic DNA restriction fragments, wherein the composition of therestriction fragment mixture depends on the methylation pattern of thegenomic DNA; b) coupling the amplified fragments with a detectable labelto provide a labeled fragment amplificate; c) contacting the labeledfragment amplificate with at least one DNA microarray having a pluralityof different nucleic acids assigned to different respective arraylocations, wherein hybridization of amplificate fragments with thecorresponding assigned nucleic acids takes place under definablestringency, and wherein the assigned nucleic acids are specific forgenomic fragments obtainable by cutting with the restriction enzymesused in a); d) detecting the signal intensities derived from hybridizedlabeled amplificate fragments using a suitable detection method; and e)preprocessing of the detected signal intensities, wherein thepreprocessing comprises Log-Transformation, and further comprises atleast one selected from the group consisting of: Quantile Normalization;Baseline Shift; and f) generating a representative value for the signalintensity values of a set of nucleic acids immobilized on the DNAmicroarray, wherein determining a DNA methylation pattern is provided.8. A method for determining a DNA methylation pattern, comprising: a)obtaining a solution comprising a sized-biased amplified mixture ofgenomic DNA restriction fragments, wherein the composition of therestriction fragment mixture depends on the methylation pattern of thegenomic DNA; b) coupling the amplified fragments with a detectable labelto provide a labeled fragment amplificate; c) contacting the labeledfragment amplificate with at least one DNA microarray having a pluralityof different nucleic acids assigned to different respective arraylocations, wherein hybridization of amplificate fragments with thecorresponding assigned nucleic acids takes place under definablestringency, wherein the assigned nucleic acids are specific for genomicfragments obtainable by cutting with the restriction enzymes used in a),and wherein the array exclusively comprises oligonucleotide sequenceswhich hybridize in constant to each other defined distances on thecorresponding analyzed complementary DNA, providing a tiling array; andd) detecting the label of the hybridized fragment amplificate using asuitable detection method, wherein determination of a hybridizationpattern of the assigned array locations is afforded to further afforddetermination of the genomic DNA methylation pattern.
 9. The method ofany one of claims 1 through 8, wherein defining the sequences of theassigned oligonucleotides comprises: a) testing a genome for firstpartial sequences that are bordered by restriction sites ofnon-methylation-specific restriction enzymes, and that have a length ofabout 100 to about 1,200 base pairs, and thereby selecting said firstpartial sequences; b) excluding from the selected first partialsequences, sequences that comprise more than about 50% repeat sequences,or more than about 20% repeat sequences, thereby selecting secondpartial sequences; and c) selecting, arbitrarily or otherwise,oligonucleotide sequences from the second partial sequences, thecorresponding oligonucleotides to be immobilized on the microarray. 10.The method of any one of claims 1 through 8, wherein at least onemethylation-specific restriction enzyme is used for generating asize-biased amplified mixture of genomic DNA restriction fragments, andwherein defining the sequences of the assigned oligonucleotidescomprises: a) testing a genome for first partial sequences that arebordered by restriction sites of non-methylation-specific restrictionenzymes, and that have a length of about 100 to about 1,200 base pairs,and thereby selecting said first partial sequences; b) excluding fromthe selected first partial sequences, sequences that comprise more thanabout 50% repeat sequences, or more than about 20% repeat sequences,thereby selecting second partial sequences; c) testing the selectedsecond partial sequences for the presence of at least one restrictionsite of at least one methylation-specific restriction enzyme, andselecting third partial sequences having such sites; and d) selecting,arbitrarily or otherwise, oligonucleotide sequences from the thirdpartial sequences, the corresponding oligonucleotides to be immobilizedon the microarray.
 11. The method of any one of claims 1 through 8,wherein defining the sequences of the assigned oligonucleotidescomprises: a) testing a genome for first partial sequences that arebordered by restriction sites of methylation-specific restrictionenzymes, and that have a length of about 100 to about 1,200 base pairs,and thereby selecting said first partial sequences; b) excluding fromthe selected first partial sequences, sequences that comprise more thanabout 50% repeat sequences, or more than about 20% repeat sequences,thereby selecting second partial sequences sequences; and c) selecting,arbitrarily or otherwise, oligonucleotide sequences from the secondpartial sequences, the corresponding oligonucleotides to be immobilizedon the microarray.
 12. The method of any one of claims 1 through 8,wherein defining the sequences of the assigned oligonucleotidescomprises: a) testing a genome for first partial sequences that arebordered by restriction sites of at least one first restriction enzymeused for fragmentation, and that have a length of about 100 to about1,200 base pairs, and thereby selecting said first partial sequences; b)excluding from the selected first partial sequences, sequences thatcomprise more than about 50% repeat sequences, or more than about 20%repeat sequences, thereby selecting second partial sequences; c) testingthe selected second partial sequences for the presence of restrictionsites of at least one second restriction enzyme used for fragmentationand thereby selecting third partial sequences that comprise suchrestriction sites; and d) selecting, arbitrarily or otherwise,oligonucleotide sequences from the third partial sequences, thecorresponding oligonucleotides to be immobilized on the microarray. 13.The method of any one of claims 1 through 8, comprising: a) digestingthe DNA with at least one non-methylation-specific restriction enzyme,wherein the DNA is cut at corresponding restriction sites; b)selectively depleting DNA fragments less than about 50 bases in lengthfrom the digested DNA); c) ligating adaptors to the depleted DNA; d)digesting the depleted DNA with at least one methylation-specificrestriction enzyme, wherein the depleted DNA is cut at correspondingunmethylated restriction sites to provide a further digested DNA; and e)amplifying the further digested DNA, using primer mediated-amplificationof adapter-ligated fragments to provide a size biased fragmentamplificate; or comprising: a) digesting the DNA with at least onenon-methylation-specific restriction enzyme, wherein the DNA is cut atcorresponding restriction sites; b) selectively depleting DNA fragmentsless than about 50 bases in length from the digested DNA); c) ligatingadaptors to the depleted DNA; and d) amplifying the adapter-ligated DNA,using primer mediated-amplification of adaptor-ligated fragments toprovide a size biased fragment amplificate.
 14. The method of claim 1,wherein the enrichment comprises use of a) at least one of MeCP2, MBD1,MBD2, MBD4, Kaiso, or suitable domains of these proteins; b) MBD-columnchromatography, comprising a combination of enrichment of non-methylatedDNA by binding to a column specific for non-methylated DNA, withenrichment of methylated DNA by binding to a column specific formethylated DNA; and at least one of c) antibodies specific for at leastone of MeCP2, MBD1, MBD2, MBD4, Kaiso or one or more domains thereof;and d) methylation-specific antibodies.
 15. The method of claim 6,comprising, generating two corresponding types of DNA samples from eachof a test sample and a reference sample, wherein generating the firsttype of sample comprises a complexity reduction of genomic DNA that isindependent of the genomic DNA methylation pattern, wherein generatingthe second type of sample comprises a first methylation-unspecificrestriction enzyme digestion and a second methylation-specificrestriction enzyme digestion; deducing copy-number variations bycomparison of detected signal intensities of the first type of DNAfragment samples of the test sample with the detected signal intensitiesof the first type of DNA fragment samples of the reference sample; anddeducing methylation changes by comparison of detected signalintensities of the second type of DNA fragment samples of the testsample with the detected signal intensities of the second type of DNAfragment samples of the reference sample.
 16. The method of claim 6,comprising: generating two corresponding types of DNA fragment samplesfrom each of a test sample and a reference sample, wherein generatingthe first type of sample comprises a first methylation-unspecificrestriction enzyme digestion and a second methylation-specificrestriction enzyme digestion, and wherein generating the second type ofsample comprises a methylation-specific restriction enzyme digestion;deducing an alteration in DNA methylation by comparison of hybridizationsignal intensities of the first type of DNA fragments of the test samplewith those of a reference sample, or by comparison of hybridizationsignal intensities of the second type of DNA fragments of the testsample with those of the reference sample, or both; and deducing acopy-number variation by comparison of hybridization signal intensity ofthe first type of DNA fragments of a test sample with those derived fromthe reference sample, and by comparison of hybridization signalintensity of the second type of DNA fragments of the test samples withthose of the reference sample.
 17. The method of claim 6, comprising:generating, by enrichment of methylated DNA, DNA fragments from a testsample, from a completely methylated aliquot of a reference sample, andfrom a completely unmethylated aliquot of said reference sample; andobtaining a value represented by the quotient of the difference of thehybridization signal intensity of the test sample and the hybridizationsignal intensity of the completely unmethylated reference sample to thesignal difference of the completely methylated reference sample and thecompletely unmethylated reference sample; wherein an increase of thecopy-number of the analyzed genomic region in the test sample is deducedwhere quotient values are larger than
 1. 18. The method of claim 6,comprising: generating, by enrichment of unmethylated DNA, DNA fragmentsfrom a test sample, from a completely methylated aliquot of a referencesample, and from a completely unmethylated aliquot of said referencesample; and obtaining a value represented by the quotient of thedifference of the signal intensity of the test sample and the signalintensity of the completely methylated reference sample to the signaldifference of the completely unmethylated reference sample and thecompletely methylated reference sample, wherein an increase of thecopy-number of the analyzed genomic region in the test sample is deducedwhere quotient values are larger than
 1. 19. The method of claim 6,wherein generation of the sized-biased amplified mixture comprises anenrichment step using proteins that bind methylation-specifically to theDNA.
 20. The method of claim 6, wherein generation of the sized-biasedamplified mixture comprises the use of a triplex-forming molecule, whichwhen brought in contact with the DNA, distinguishes between methylatedand non-methylated DNA.
 21. The method of claim 6, wherein generation ofthe sized-biased amplified mixture comprises use of the MS AP-PCRmethod.